Distribution of Aromatic Amino Acid Residues in Substrate-Binding Regions Modulates Substrate Specificity of Microbial Debranching Enzymes.

Debranching enzymes (DBEs) directly hydrolyze α-1,6-glucosidic linkages in glycogen, starch, and related polysaccharides, making them important in the starch processing industry. However, the ambiguous substrate specificity usually restricts synergistic catalysis with other amylases for improving starch utilization. Herein, a glycogen-debranching enzyme from Saccharolobus solfataricus (SsGDE) and two isoamylases from Pseudomonas amyloderamosa (PaISO) and Chlamydomonas reinhardtii (CrISO) were used to investigate the molecular mechanism of substrate specificity. Along with the structure-based computational analysis, the aromatic residues in the substrate-binding region of DBEs played an important role in binding substrates. The aromatic residues in SsGDE appeared clustered, contributing to a small substrate-binding region. In contrast, the aromatic residues in isoamylase were distributed dispersedly, forming a large active site. The distinct characteristics of substrate-binding regions in SsGDE and isoamylase might explain their substrate preferences for maltodextrin and amylopectin, respectively. By modulating the substrate-binding region of SsGDE, variants Y323F and V375F were obtained with significantly enhanced activities, and the activities of Y323F and V375F increased by 30 and 60% for amylopectin, and 20 and 23% for DE4 maltodextrin, respectively. This study revealed the molecular mechanisms underlying the substrate specificity for SsGDE and isoamylases, providing a route for engineering enzymes to achieve higher catalytic performance.

Mutation in BEIIb mitigates the negative effect of the mutation in ISA1 on grain filling and amyloplast formation in rice.

KEY MESSAGE: Mutation of the BEIIb gene in an isa1 mutant background mitigates the negative effect of the ISA1 mutation on grain filling, and facilitates recovery of amyloplast formation in rice endosperm. In this study, the effect of branching enzyme IIb and isoamylase 1 deficiency on starch properties was demonstrated using high resistant starch rice lines, Chikushi-kona 85 and EM129. Both lines harbored a mutation in the BEIIb and ISA1 genes and showed no BEIIb and ISA1 activity, implying that both lines are beIIb isa1 double mutants. The amylopectin long chain and apparent amylose content of both mutant lines were higher than those of the wild-type. While both mutants contained loosely packed, round starch grains, a trait specific to beIIb mutants, they also showed collapsed starch grains at the center of the endosperm, a property specific to isa1 mutants. Furthermore, beIIb isa1 double mutant F2 lines derived from a cross between Chikushi-kona 85 and Nishihomare (wild-type cultivar) showed significantly heavier seed weight than the beIIb and isa1 single mutant lines. These results suggest that co-occurrence of beIIb and isa1 mutant alleles in a single genetic background mitigates the negative effect of the isa1 allele on grain filling, and contributes to recovery of the amyloplast formation defect in the isa1 single mutant.

Foliar Application of dsRNA Targeting Endogenous Potato (Solanum tuberosum) Isoamylase Genes ISA1, ISA2, and ISA3 Confers Transgenic Phenotype.

Isoamylase (ISA) is a debranching enzyme found in many plants, which hydrolyzes (1-6)-α-D glucosidic linkages in starch, amylopectin, and ß-dextrins, and is thought to be responsible for starch granule formation (ISA1 and ISA2) and degradation (ISA3). Lipid-modified PEI (lmPEI) was synthesized as a carrier for long double-stranded RNA (dsRNA, 250-bp), which targets the three isoamylase isoforms. The particles were applied to the plant via the foliar spray and were differentially effective in suppressing the expressions of ISA1 and ISA2 in the potato leaves, and ISA3 in the tubers. Plant growth was not significantly impaired, and starch levels in the tubers were not affected as well. Interestingly, the treated plants had significantly smaller starch granule sizes as well as increased sucrose content, which led to an early sprouting phenotype. We confirm the proposal of previous research that an increased number of small starch granules could be responsible for an accelerated turnover of glucan chains and, thus, the rapid synthesis of sucrose, and we propose a new relationship between ISA3 and the starch granule size. The implications of this study are in achieving a transgenic phenotype for endogenous plant genes using a systemic, novel delivery system, and foliar applications of dsRNA for agriculture.

Characterization and differential expression of sucrose and starch metabolism genes in contrasting chickpea (Cicer arietinum L.) genotypes under low temperature.

Low temperature (LT) causes significant yield losses in chickpea (Cicer arietinum L.). The sucrose starch metabolism is associated with abiotic-stress tolerance or sensitivity in plants. The changes in sugars and starch contents under LT in chickpea have already been studied, however, no information is available on LT-induced alterations in transcription of carbohydrate metabolic pathway genes in chickpea. To understand the differences in the regulation of sucrose and starch metabolism under LT, the expression of sucrose and starch metabolism genes was studied in leaves of cold-sensitive (GPF2) and cold-tolerant (ICC 16349) chickpea genotypes. The mRNA sequences of chickpea genes were retrieved from the public databases followed by confirmation of identity and characterization. All the genes were functional in chickpea. Between the two paralogues of cell wall invertase, cell wall invertase 3×2 (CWINx2) was the truncated version of cell wall invertase 3×1 (CWINx1) with the loss of 241 bases in the mRNA and 67 amino acids at N terminal of the protein. Comparison of expression of the genes between control (22°C day / 16°C night) and LT treated (4°C; 72 h) plants revealed that granule bound starch synthase 2 (GBSS2) and ß-amylase 3 (BAM3) were upregulated in ICC 16349 whereas sucrose phosphate synthase 2 (SPS2), CWINx1, CWINx2 and ß-amylase 1 (BAM1) were downregulated. In contrast to this, SPS2, CWINx1, CWINx2 and BAM1 were upregulated and GBSS2 downregulated in GPF2 under LT. The gene expression data suggested that UGPase, CWINs, GBSS2 and BAM3 are important components of cold-tolerance machinery of chickpea.

Improvement of starch films for food packaging through a three-principle approach: Antioxidants, cross-linking and reinforcement.

This study uses sunflower hulls, a by-product from the sunflower snack industry, to recover both, valuable phenolic compounds and cellulose fibers, for the production of antioxidant reinforced starch films as potential food packaging material. The phenolic extract provided antioxidant properties to the films with EC50 values of 89 mg film/mg DPPH. The cellulose fibers reinforced the starch films with a threefold increase in Young´s modulus. Furthermore, citric acid was added to induce cross-linking of the starch polymers and improve film integrity. The addition of citric acid induced both, starch polymer hydrolysis and cross-linking, seen in a shift in chain-length distribution after debranching with iso-amylase. This is the first study that focuses on a three-principle approach to improve edible starch films, and follows UN goals on sustainability to reduce waste and increase value in by-products as a step forward to functionalize packaging material.

The relationship between the expression pattern of starch biosynthesis enzymes and molecular structure of high amylose maize starch.

Two high amylose (HAM) inbred lines with apparent amylose contents of 55 % and 62 %, respectively, were selected to explore the relationship between molecular structure and gene expression of starch-synthase involved enzymes. GPC analysis of debranched starches showed that the HAM starches (HAMSs) had shorter amylose chains and longer amylopectin chains than normal maize starch (NMS). FACE analysis showed that these HAMSs had a higher content of amylopectin chains of DP > 21. Quantitative Real-Time PCR analysis showed that the HAM lines had specifically low expression of the starch branching enzyme IIb (SBEIIb), and the starch synthase IIIa (SSIIIa) homologue, and high expression of the isoamylase 2 (ISA2), potentially suppressing the generation of amylopectin molecules through deficient branching and excessive debranching process, thereby increasing the relative amylose content. A high expression of GBSS1 was potentially associated with increased short amylose chain lengths in HAMSs.

Efficient secretory production of large-size heterologous enzymes in Bacillus subtilis: A secretory partner and directed evolution.

Secretory production of recombinant proteins provides a simple approach to the production and purification of target proteins in the enzyme industry. We developed a combined strategy for the secretory production of three large-size heterologous enzymes with a special focus on 83-kDa isoamylase (IA) from an archaeon Sulfolobus tokodaii in a bacterium Bacillus subtilis. First, a secretory protein of the B. subtilis family 5 glycoside hydrolase endoglucanase (Cel5) was used as a fusion partner, along with the NprB signal peptide, to facilitate secretory production of IA. This secretory partner strategy was effective for the secretion of two other large enzymes: family 9 glycoside hydrolase from Clostridium phytofermentas and cellodextrin phosphorylase from Clostridium thermocellum. Second, the secretion of Cel5-IA was improved by directed evolution with two novel double-layer Petri-dish-based high-throughput screening (HTS) methods. The high-sensitivity HTS relied on the detection of high-activity Cel5 on the carboxymethylcellulose/Congo-red assay. The second modest-sensitivity HTS focused on the detection of low-activity IA on the amylodextrin-I2 assay. After six rounds of HTS, a secretory Cel5-IA level was increased to 234 mg/L, 155 times the wild-type IA with the NprB signal peptide only. This combinatory strategy could be useful to enhance the secretory production of large-size heterologous proteins in B. subtilis.

Bio characterization of purified isoamylase from Rhizopus oryzae.

Extracellular isoamylase produced by Rhizopus oryzae PR7 MTCC 9642 in in Erlenmeyer flasks was purified by ultrafiltration and by two steps of Superose 6 C-10/300GL gel chromatography. The enzyme molecule was found to be a monomer with molecular weight of 68 kDa.The purified isoamylase showed optimum activity at pH 5.5 and temperature 55 °C. The catalytic activity was found to remain stable at a broad range of pH (4-8) and could show remarkable thermo resistance specially in presence of exogenous thiols. The noteworthy enhancement of activity in presence of Mn2+ indicated its role as enzyme cofactor while thermos and chemostability in presence of exogenous thiols indicated the presence of disulfide linkage at active site of the enzyme. Both in vitro study and doking analysis indicated the highest affinity of the isoamylase of R. oryzae PR7 toward glycogen and the enzyme exhibited Km and Vmax values of 0.38 mg/mL and 6.65 mM/min/mL, respectively. Purified debranching amylolytic enzyme from R. oryzae PR7 has potential for the study of glycogen and starch structure and industrial application in combination with other amylolytic enzymes. The rapid, convenient, relatively simple purification process and other functional attributes of the enzyme made it competent to be employed for industrial utilization.

Effects of partial debranching and storage temperature on recrystallization of waxy maize starch.

Waxy maize starch was debranched using an isoamylase (hydrolysis for 24 h or 48 h), and then the debranched starches were recrystallized for 96 h at different temperatures (4, 25, and 50 °C). The structural and crystalline characteristics of the recrystallized samples were analyzed. The longer debranching time (48 h) produced the smaller chains containing more of linear dextrins. Crystallinity and thermal stability were much higher for the starch crystals prepared with the higher level of debranching. The starch chains recrystallized at 4 and 25 °C were recovered at 84-93% yield with B-type crystals whereas those at 50 °C induced a lower yield (11-31%) with A-type crystals. The slow recrystallization (50 °C) induced the selective association of the long chains which led to the formation of A-type crystals having a good thermal stability. The recrystallization temperature was a determining factor for the crystalline arrangement of debranched starch chains.

CO2-Responsive CCT Protein Stimulates the Ectopic Expression of Particular Starch Biosynthesis-Related Enzymes, Which Markedly Change the Structure of Starch in the Leaf Sheaths of Rice.

CO2-responsive CCT protein (CRCT) is suggested to be a positive regulator of starch biosynthesis in the leaf sheaths of rice, regulating the expression levels of starch biosynthesis-related genes. In this study, the effects of CRCT expression levels on the expression of starch biosynthesis-related enzymes and the quality of starch were studied. Using native-PAGE/activity staining and immunoblotting, we found that the protein levels of starch synthase I, branching enzyme I, branching enzyme IIa, isoamylase 1 and phosphorylase 1 were largely correlated with the CRCT expression levels in the leaf sheaths of CRCT transgenic lines. In contrast, the CRCT expression levels largely did not affect the expression levels and/or activities of starch biosynthesis-related enzymes in the leaf blades and endosperm tissues. The analysis of the chain-length distribution of starch in the leaf sheaths showed that short chains with a degree of polymerization from 5 to 14 were increased in the overexpression lines but decreased in the knockdown lines. The amylose content of starch in the leaf sheath was greatly increased in the overexpression lines. In contrast, the molecular weight of the amylopectin of starch in the leaf sheath of overexpression lines did not change compared with those of the non-transgenic rice. These results suggest that CRCT can control the quality and the quantity of starch in the leaf sheath by regulating the expression of particular starch biosynthesis-related enzymes.

Phosphorus Enhances Photosynthetic Storage Starch Production in a Green Microalga (Chlorophyta) Tetraselmis subcordiformis in Nitrogen Starvation Conditions.

Microalgae are potential starch producers as alternatives to agricultural crops. This study disclosed the effects and mechanism of phosphorus availability exerted on storage starch production in a starch-producing microalga Tetraselmis subcordiformis in nitrogen starvation conditions. Excessive phosphorus supply facilitated starch production, which differed from the conventional cognition that phosphorus would inhibit transitory starch biosynthesis in plants. Phosphorus enhanced energy utilization efficiency for biomass and storage starch production. ADP-glucose pyrophosphorylase (AGPase), conventionally known to be critical for starch biosynthesis, was negatively correlated to storage starch biosynthesis. Excessive phosphorus supply maintained large cell volumes, enhanced activities of starch phosphorylases (SPs) along with branching enzymes and isoamylases, and increased phosphoenolpyruvate and trehalose-6-phosphate levels to alleviate the inhibition of high phosphate availability to AGPase, all of which improved starch production. This work highlighted the importance of phosphorus in the production of microalgal starch and provided further evidence for the SP-based storage starch biosynthesis pathway.

Endosperm sugar accumulation caused by mutation of PHS8/ISA1 leads to pre-harvest sprouting in rice.

Pre-harvest sprouting (PHS) is an unfavorable trait in cereal crops that could seriously decrease grain yield and quality. Although some PHS-associated quantitative trait loci or genes in cereals have been reported, the molecular mechanism underlying PHS remains largely elusive. Here, we characterized a rice mutant, phs8, which exhibits PHS phenotype accompanied by sugary endosperm. Map-based cloning revealed that PHS8 encodes a starch debranching enzyme named isoamylase1. Mutation in PHS8 resulted in the phytoglycogen breakdown and sugar accumulation in the endosperm. Intriguingly, with increase of sugar contents, decreased expression of OsABI3 and OsABI5 as well as reduced sensitivity to abscisic acid (ABA) were found in the phs8 mutant. Using rice suspension cell system, we confirmed that exogenous sugar is sufficient to suppress the expression of both OsABI3 and OsABI5. Furthermore, overexpression of OsABI3 or OsABI5 could partially rescue the PHS phenotype of phs8. Therefore, our study presents important evidence supporting that endosperm sugar not only acts as an essential energy source for seed germination but also determines seed dormancy and germination by affecting ABA signaling.

Heterologous co-expression in E. coli of isoamylase genes from cassava Manihot esculenta Crantz 'KU50' achieves enzyme-active heteromeric complex formation.

KEY MESSAGE: Cloning of two isoamylase genes, MeISA1 and MeISA2, from cassava (Manihot esculenta Crantz) tubers, accompanied by their co-expression in E. coli demonstrates a requirement for heteromeric complex formation to achieve debranching activity. Starch debranching enzyme (DBE) or isoamylase (ISA) (EC.3.2.1.68), an important enzyme in starch metabolism, catalyses the hydrolysis of α-1,6 glycosidic linkages of amylopectin. Isoforms of ISAs have been reported in higher plants and algae (Fujita et al. in Planta 208:283-293, 1999; Hussain et al. in Plant Cell 15:133-149, 2003; Ishizaki et al. in Agric Biol Chem 47:771-779, 1983; Mouille et al. in Plant Cell 8:1353-1366, 1996). In the current work, cassava ISA genes were isolated from cDNA generated from total RNA from tubers of Manihot esculanta Crantz cultivar KU50. MeISA1 and MeISA2 were successfully amplified and cloned into a pETDuet1 vector. The putative MeISA1 and MeISA2 proteins comprised 763 and 882 amino acids, with substantial similarity to StISA1 and StISA2 from potato (84.4% and 68.9%, respectively). Recombinant MeISA1 and MeISA2 were co-expressed in Escherichia coli SoluBL21 (DE3). HistrapTM-Purified rMeISA1 and rMeISA2 showed approximate molecular weights of 87 and 99 kDa, respectively, by SDS-PAGE. Debranching activity was only detectable in the column fractions where both recombinant ISA isoforms were present. The heteromeric DBE from crude extracts of 4-5 h induced cultures analysed by gel filtration chromatography and western blot showed combinations of rMeISA1 and rMeISA2 at ratios of 1:1 to 4:1. Pooled fractions with DBE activity were used for enzyme characterisation, which showed that the enzyme was specific for amylopectin, with optimum activity at 37 °C and pH 7.0. Enzyme activity was enhanced by Co2+, Mg2+ and Ca2+, but was strongly inhibited by Cu2+. Debranched amylopectin products showed chain length distributions typical of plant DBE.

Homologs of PROTEIN TARGETING TO STARCH Control Starch Granule Initiation in Arabidopsis Leaves.

The molecular mechanism that initiates the synthesis of starch granules is poorly understood. Here, we discovered two plastidial proteins involved in granule initiation in Arabidopsis thaliana leaves. Both contain coiled coils and a family-48 carbohydrate binding module (CBM48) and are homologs of the PROTEIN TARGETING TO STARCH (PTST) protein; thus, we named them PTST2 and PTST3. Chloroplasts in mesophyll cells typically contain five to seven granules, but remarkably, most chloroplasts in ptst2 mutants contained zero or one large granule. Chloroplasts in ptst3 had a slight reduction in granule number compared with the wild type, while those of the ptst2 ptst3 double mutant contained even fewer granules than ptst2 The ptst2 granules were larger but similar in morphology to wild-type granules, but those of the double mutant had an aberrant morphology. Immunoprecipitation showed that PTST2 interacts with STARCH SYNTHASE4 (SS4), which influences granule initiation and morphology. Overexpression of PTST2 resulted in chloroplasts containing many small granules, an effect that was dependent on the presence of SS4. Furthermore, isothermal titration calorimetry revealed that the CBM48 domain of PTST2, which is essential for its function, interacts with long maltooligosaccharides. We propose that PTST2 and PTST3 are critical during granule initiation, as they bind and deliver suitable maltooligosaccharide primers to SS4.

ZmMYB14 is an important transcription factor involved in the regulation of the activity of the ZmBT1 promoter in starch biosynthesis in maize.

The biosynthesis of starch is a complex process that depends on the regulatory mechanisms of different functional enzymes, and transcriptional regulation plays an important role in this process. Brittle 1, encoded by BT1, is a transporter of adenosine diphosphate-glucose, which plays an important role in the biosynthesis of starch in the endosperm of cereals. Here, we report that the promoter (pZmBT1) of the maize BT1 homolog, ZmBT1, contains an MBSI site (TAACTG), which is important for its activity. Moreover, high expression level of the gene for ZmMYB14 transcription factor was observed in the maize endosperm; its expression pattern was similar to those of the starch synthesis-related genes in maize seeds. ZmMYB14 is a typical 2R-MYB transcription factor localized in the nucleus and possessed transcriptional activation activity. ZmMYB14 could bind to the region of pZmBT1 from -280 to -151 bp and promote its activity through the TAACTG site. It was also observed to promote the activity of pZmSh2, pZmBt2, pZmGBSSI, pZmSSI, and pZmSBE1 in the maize endosperm in transient gene overexpression assays. Furthermore, ZmMYB14 was also shown to bind directly to the promoters of six starch-synthesizing genes, ZmGBSSI, ZmSSI, ZmSSIIa, ZmSBE1, ZmISA1, and ZmISA2 in yeast. These findings indicate that ZmMYB14 functions as a key regulator of ZmBT1 and is closely related to the biosynthesis of starch. Our results provide crucial information related to the regulation of starch biosynthesis in maize and would be helpful in devising strategies for modulating starch production in maize endosperm.

Simultaneous silencing of isoamylases ISA1, ISA2 and ISA3 by multi-target RNAi in potato tubers leads to decreased starch content and an early sprouting phenotype.

Isoamylases hydrolyse (1-6)-alpha-D-glucosidic linkages in starch and are involved in both starch granule formation and starch degradation. In plants, three isoamylase isoforms with distinct functions in starch synthesis (ISA1 and ISA2) and degradation (ISA3) have been described. Here, we created transgenic potato plants with simultaneously decreased expression of all three isoamylases using a chimeric RNAi construct targeting all three isoforms. Constitutive expression of the hairpin RNA using the 35S CaMV promoter resulted in efficient silencing of all three isoforms in leaves, growing tubers, and sprouting tubers. Neither plant growth nor tuber yield was effected in isoamylase-deficient potato lines. Interestingly, starch metabolism was found to be impaired in a tissue-specific manner. While leaf starch content was unaffected, tuber starch was significantly reduced. The reduction in tuber starch content in the transgenic plants was accompanied by a decrease in starch granules size, an increased sucrose content and decreased hexose levels. Despite the effects on granule size, only little changes in chain length composition of soluble and insoluble glucose polymers were detected. The transgenic tubers displayed an early sprouting phenotype that was accompanied by an increased level of sucrose in parenchyma cells below the outgrowing bud. Since high sucrose levels promote sprouting, we propose that the increased number of small starch granules may cause an accelerated turnover of glucan chains and hence a more rapid synthesis of sucrose. This observation links alterations in starch structure/degradation with developmental processes like meristem activation and sprout outgrowth in potato tubers.

Investigation of debranching pattern of a thermostable isoamylase and its application for the production of resistant starch.

Debranching enzymes contribute to the enzymatic production of resistant starch (RS) by reducing substrate molecular weight and increasing amylose yield. In the present study, the action pattern of a thermostable isoamylase-type debranching enzyme on different types of starch was investigated. The molecular weight distribution, glycosidic bond composition and contents of oligosaccharides released were monitored by various liquid chromatography techniques and nuclear magnetic resonance spectroscopy (NMR). These analyses showed that the isoamylase could specifically and efficiently attack α-1,6-glucosidic linkages at branch points, leaving the amylose favored by other amylolytic enzymes. Its ability to attack side chains composed of 1-3 glucose residues differentiates it from other isoamylases, a property which is also ideal for the RS preparation process. The enzyme was used as an auxiliary enzyme in the hydrolytic stage. The highest RS yield (53.8%) was achieved under the optimized conditions of 70 °C and pH 5.0, using 7 U isoamylase per g starch and 2 NU amylase per g starch. These data also help us better understand the application of isoamylase for preparation of other products from highly branched starch materials.

The buffering capacity of stems: genetic architecture of nonstructural carbohydrates in cultivated Asian rice, Oryza sativa.

Harnessing stem carbohydrate dynamics in grasses offers an opportunity to help meet future demands for plant-based food, fiber and fuel production, but requires a greater understanding of the genetic controls that govern the synthesis, interconversion and transport of such energy reserves. We map out a blueprint of the genetic architecture of rice (Oryza sativa) stem nonstructural carbohydrates (NSC) at two critical developmental time-points using a subpopulation-specific genome-wide association approach on two diverse germplasm panels followed by quantitative trait loci (QTL) mapping in a biparental population. Overall, 26 QTL are identified; three are detected in multiple panels and are associated with starch-at-maturity, sucrose-at-maturity and NSC-at-heading. They tag OsHXK6 (rice hexokinase), ISA2 (rice isoamylase) and a tandem array of sugar transporters. This study provides the foundation for more in-depth molecular investigation to validate candidate genes underlying rice stem NSC and informs future comparative studies in other agronomically vital grass species.

Effect of different amylases on the utilization of cornstarch in broiler chickens.

This study compared the effect of different amylases on the utilization of cornstarch in broiler chickens fed a corn-based diet. Three-day-old Arbor Acres plus male chickens were randomly divided into 7 treatments and fed a diet supplemented with different sources and concentrations of amylase: 1,500 U/kg and 3,000 U/kg α-1,4 amylase from Aspergillus oryzae (α-amylase A); 480 U/kg and 960 U/kg α-1,4 amylase from Bacillus subtilis (α-amylase B); 200 U/kg and 400 U/kg α-1,6 isoamylase from B. subtilis; and the control. The experimental period comprised 11 d, during which performance, nutrient digestibility, digestive enzyme activity, intestinal morphology, and glucose transporter transcription of the chickens were evaluated. The results indicated that 1,500 U/kg α-amylase improved the digestibility of energy and decreased the feed conversion rate compared to α-1,6 isoamylase (P < 0.05). Supplemental 400 U/kg α-1,6 isoamylase decreased ileal digestibility of amylopectin and total starch (P < 0.05) compared to 200 U/kg α-1,6 isoamylase, α-amylase A, α-amylase B, and the control (P < 0.05). Supplemental α-1,6 isoamylase decreased (P < 0.05) insulin content. Supplemental 3,000 U/kg α-amylase A and α-1,6 isoamylase increased (P < 0.05) the relative weight of the liver. In addition, 3,000 U/kg α-amylase A, 480 U/kg α-amylase B, and α-1,6 isoamylase decreased the V:C in the duodenum and ileum. α-amylase A increased sucrase activity in the jejunum (P < 0.05), whereas 400 U/kg α-1,6 isoamylase reduced maltase activity in the duodenum (P < 0.05). Furthermore, 3,000 U/kg α-amylase A and α-amylase B decreased (P < 0.05) sodium/glucose cotransporter 1 (SGLT1) mRNA expression in the duodenum and jejunum. However, 200 U/kg α-1,6 isoamylase increased glucose transporter 2 (GLUCT2) in the duodenum (P < 0.05). These results suggest that exogenous amylase affects the digestibility of starch by affecting disaccharidase activity in the intestine, nutrient requirements for intestinal maintenance by the V:C, and nutrient absorption and metabolism via GLU transporter mRNA expression. Different sources and concentrations of amylases had varying effects on broilers.