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PubMed2002

Effect of C-domain N-glycosylation and deletion on rat pancreatic alpha-amylase secretion and activity.

Even though all animal alpha-amylases include glycosylation sequons (Asn-Xaa-Thr/Ser) in their sequences, amylases purified from natural sources are not quantitatively glycosylated. When wild-type rat pancreatic alpha-amylase, which contains two glycosylation sequons, was expressed in animal cell lines the protein displayed a very low rate of glycosylation (approx. 2%), even after Brefeldin A treatment to increase the contact with the glycosylation machinery. Site-directed mutagenesis of the first glycosylation sequon (Asn(410)-->Gln) resulted in 90% of the protein being glycosylated at the second glycosylation sequon (Asn(459)). Mutation of the second sequon completely inhibited glycosylation. In order to ascertain if the interference in the glycosylation of Asn(459) that was eliminated by the Asn(410)-->Gln mutation could be due to the position of the asparagine residue in the Cys(448)-Cys(460) disulphide bridge, these cysteine residues were mutated to serine residues. The resulting mutant was found to be 100% glycosylated. All mutants with mutations in the C-domain had specific activities identical to that of the wild-type enzyme, indicating that enzymic activity is independent of the structure and modification of the C-terminal domain. To further test the independence of the C-domain with respect to the two N-terminal domains of the protein, which harbour the catalytic site, the last seven of the ten beta\beta-strands that make up the beta-sandwich configuration of the domain were deleted. The truncated protein was not secreted from cells and all enzyme activity was destroyed. These observations show that Asn(459) is the only site that can be glycosylated in wild-type amylase, and confirm the relative independence of the C-terminal domain of alpha-amylase with respect to enzyme activity. In addition, they also establish that the C-terminal domain is absolutely essential for the correct post-translational folding of the enzyme that is responsible for its activity and allows for its secretion.

Animals↗
PubMed2003

Effect of calcium ions on the irreversible denaturation of a recombinant Bacillus halmapalus alpha-amylase: a calorimetric investigation.

The effect of temperature and calcium ions on the denaturation of a recombinant alpha-amylase from Bacillus halmapalus alpha-amylase (BHA) has been studied using calorimetry. It was found that thermal inactivation of BHA is irreversible and that calcium ions have a significant effect on stability. Thus an apparent denaturation temperature ( T (d)) of 83 degrees C in the presence of excess calcium ions was observed, whereas T (d) decreased to 48 degrees C when calcium was removed. The difference in thermal stability with and without calcium ions has been used to develop an isothermal titration calorimetric (ITC) procedure that allows simultaneous determination of kinetic parameters and enthalpy changes of the denaturation of calcium-depleted BHA. An activation energy E (A) of 101 kJ/mol was found for the denaturation of calcium-depleted BHA. The results support a kinetic denaturation mechanism where the calcium-depleted amylase denatures irreversibly at low temperature and if calcium ions are in excess, the amylase denatures irreversibly at high temperatures. The two denaturation reactions are coupled with the calcium-binding equilibrium between calcium-bound and -depleted amylase. A combination of the kinetic denaturation results and calcium-binding constants, determined by isothermal titration calorimetry, has been used to estimate kinetic stability, expressed in terms of the half-life of BHA as a function of temperature and free-calcium-ion concentration. Thus it is estimated that the apparent E (A) can be increased to approx. 123 kJ/mol by increasing the free-calcium concentration.

Bacillus↗
PubMed1988

Evidence against direct involvement of cyclic AMP-dependent protein phosphorylation in the exocytosis of amylase.

To examine whether or not the activation of cyclic AMP-dependent protein kinase is coupled to the exocytosis of amylase from rat parotid cells, the effect of protein kinase inhibitors on amylase release and protein phosphorylation was studied. A membrane-permeable inhibitor of cyclic AMP-dependent protein kinase, N-[2-(methylamino)ethyl]-5-isoquinolinesulphonamide (H-8), and peptide fragments of the heat-stable protein kinase inhibitor [PKI-(5-24)-peptide and PKI-(14-24)-amide] strongly inhibited cyclic AMP-dependent protein kinase activity in the cell homogenate. However, H-8 had no inhibitory effect on amylase release from either intact or saponin-permeabilized parotid cells stimulated by isoproterenol or cyclic AMP. Moreover, PKI-(5-24)-peptide and PKI-(14-24)-amide did not inhibit cyclic AMP-evoked amylase release from saponin-permeabilized cells, whereas cyclic AMP-dependent phosphorylations of 21 and 26 kDa proteins in intact or permeabilized cells were markedly inhibited by these inhibitors. These results suggest that cyclic AMP-dependent protein phosphorylation is not directly involved in the exocytosis of amylase regulated by cyclic AMP.

Amylases↗
PubMed1992

Amylase release from streptolysin O-permeabilized pancreatic acinar cells. Effects of Ca2+, guanosine 5'-[gamma-thio]triphosphate, cyclic AMP, tetanus toxin and botulinum A toxin.

The molecular requirements for amylase release and the intracellular effects of botulinum A toxin and tetanus toxin on amylase release were investigated using rat pancreatic acinar cells permeabilized with streptolysin O. Micromolar concentrations of free Ca2+ evoked amylase release from these cells. Maximal release was observed in the presence of 30 microM free Ca2+. Ca(2+)-stimulated, but not basal, amylase release was enhanced by guanosine 5'-[gamma-thio]triphosphate (GTP[S]) (3-4 fold) or cyclic AMP (1.5-2 fold). Neither the two-chain forms of botulinum A toxin and tetanus toxin, under reducing conditions, nor the light chains of tetanus toxin, inhibited amylase release triggered by Ca2+, or combinations of Ca2+ + GTP[S] or Ca2+ + cAMP. The lack of inhibition was not due to inactivation of botulinum A toxin or tetanus toxin by pancreatic acinar cell proteolytic enzymes, as toxins previously incubated with permeabilized pancreatic acinar cells inhibited Ca(2+)-stimulated [3H]noradrenaline release from streptolysin O-permeabilized adrenal chromaffin cells. These data imply that clostridial neurotoxins inhibit a Ca(2+)-dependent mechanism which promotes exocytosis in neural and endocrine cells, but not in exocrine cells.

Amylases↗
PubMed1998

Cholecystokinin octapeptide inhibits Ca2+-dependent amylase secretion from permeabilized pancreatic acini by blocking the MgATP-dependent priming of exocytosis.

At present little is known about how the low-affinity cholecystokinin receptor inhibits secretagogue-stimulated amylase secretion from pancreatic acinar cells. To examine this question we have determined how cholecystokinin octapeptide (CCK8) influences Ca2+-dependent amylase secretion from alpha-toxin-permeabilized pancreatic acini. CCK8 significantly inhibited Ca2+-stimulated amylase secretion. The inhibitory actions of CCK8 were completely blocked by the addition of JMV-180, a specific antagonist for the low-affinity CCK8 receptor. Previous studies have shown that Ca2+-dependent amylase secretion from alpha-toxin-permeabilized acini has two distinct phases [Padfield and Panesar (1997) Am. J. Physiol. 36, G655-660]. There is an initial rapid phase of secretion which represents release from exocytotic sites primed by MgATP prior to permeabilization. This is followed by a slower sustained phase of secretion which, in part, reflects the MgATP-dependent repriming of the exocytotic machinery. CCK8 did not influence the initial rapid phase of the Ca2+-dependent secretory response, but inhibited the second slower sustained phase. Moreover, CCK8 was shown to inhibit the MgATP-dependent priming of exocytosis in the acini. These results indicate that the low-affinity CCK receptor blocks stimulated amylase secretion by inhibiting the MgATP-dependent repriming of exocytosis.

Adenosine Triphosphate↗
PubMed1999

An Arabidopsis gene encoding a chloroplast-targeted beta-amylase.

beta-Amylase is one of the most abundant starch degrading activities found in leaves and other plant organs. Despite its abundance, most if not all of this activity has been reported to be extrachloroplastic and for this reason, it has been assumed that beta-amylases are not involved in the metabolism of chloroplast-localized transitory leaf starch. However, we have identified a novel beta-amylase gene, designated ct-Bmy, which is located on chromosome IV of Arabidopsis thaliana. Ct-Bmy encodes a precursor protein which contains a typical N-terminal chloroplast import signal and is highly similar at the amino acid level to extrachloroplastic beta-amylases of higher plants. Expression of the ct-Bmy cDNA in E. coli confirmed that the encoded protein possesses beta-amylase activity. CT-BMY protein, synthesized in vitro, was efficiently imported by isolated pea chloroplasts and shown to be located in the stroma. In addition, fusions between the predicted CT-BMY transit peptide and jellyfish green fluorescent protein (GFP) or the entire CT-BMY protein and GFP showed accumulation in vivo in chloroplasts of Arabidopsis. Expression of the GUS gene fused to ct-Bmy promoter sequences was investigated in transgenic tobacco plants. GUS activity was most strongly expressed in the palisade cell layer in the leaf blade and in chlorenchyma cells associated with the vascular strands in petioles and stems. Histochemical staining of whole seedlings showed that GUS activity was largely confined to the cotyledons during the first 2 weeks of growth and appeared in the first true leaves at approximately 4 weeks.

Amino Acid Sequence↗
PubMed2002

Downregulation of a chloroplast-targeted beta-amylase leads to a starch-excess phenotype in leaves.

A functional screen in Escherichia coli was established to identify potato genes coding for proteins involved in transitory starch degradation. One clone isolated had a sequence very similar to a recently described chloroplast-targeted beta-amylase of Arabidopsis. Expression of the gene in E. coli showed that the protein product was a functional beta-amylase that could degrade both starch granules and solubilized amylopectin, while import experiments demonstrated that the beta-amylase was imported and processed into pea chloroplasts. To study the function of the protein in transitory starch degradation, transgenic potato plants were generated where its activity was reduced using antisense techniques. Analysis of plants reduced in the presence of this beta-amylase isoform showed that their leaves had a starch-excess phenotype, indicating a defect in starch degradation. In addition, it was shown that the antisense plants degraded only 8-30% of their total starch, in comparison with 50% in the wild type, over the dark period. This is the first time that a physiological role for a beta-amylase in plants has been demonstrated.

Chloroplasts↗
PubMed2001

Solution structure of the main alpha-amylase inhibitor from amaranth seeds.

The most abundant alpha-amylase inhibitor (AAI) present in the seeds of Amaranthus hypochondriacus, a variety of the Mexican crop plant amaranth, is the smallest polypeptide (32 residues) known to inhibit alpha-amylase activity of insect larvae while leaving that of mammals unaffected. In solution, 1H NMR reveals that AAI isolated from amaranth seeds adopts a major trans (70%) and minor cis (30%) conformation, resulting from slow cis-trans isomerization of the Val15-Pro16 peptide bond. Both solution structures have been determined using 2D 1H-NMR spectroscopy and XPLOR followed by restrained energy refinement in the consistent-valence force field. For the major isomer, a total of 563 distance restraints, including 55 medium-range and 173 long-range ones, were available from the NOESY spectra. This rather large number of constraints from a protein of such a small size results from a compact fold, imposed through three disulfide bridges arranged in a cysteine-knot motif. The structure of the minor cis isomer has also been determined using a smaller constraint set. It reveals a different backbone conformation in the Pro10-Pro20 segment, while preserving the overall global fold. The energy-refined ensemble of the major isomer, consisting of 20 low-energy conformers with an average backbone rmsd of 0.29 +/- 0.19 A and no violations larger than 0.4 A, represents a considerable improvement in precision over a previously reported and independently performed calculation on AAI obtained through solid-phase synthesis, which was determined with only half the number of medium-range and long-range restraints reported here, and featured the trans isomer only. The resulting differences in ensemble precision have been quantified locally and globally, indicating that, for regions of the backbone and a good fraction of the side chains, the conformation is better defined in the new solution structure. Structural comparison of the solution structure with the X-ray structure of the inhibitor when bound to its alpha-amylase target in Tenebrio molitor shows that the backbone conformation is only slightly adjusted on complexation, while that of the side chains involved in protein-protein contacts is similar to those present in solution. Therefore, the overall conformation of AAI appears to be predisposed to binding to its target alpha-amylase, confirming the view that it acts as a lid on top of the alpha-amylase active site.

Amaranth Dye↗
PubMed2000

Production and partial characterization of thermostable and calcium-independent alpha-amylase of an extreme thermophile Bacillus thermooleovorans NP54.

AIM: An investigation was carried out on the production of alpha-amylase by Bacillus thermooleovorans NP54, its partial purification and characterization. METHODS AND RESULTS: The thermophilic bacterium was grown in shake flasks and a laboratory fermenter containing 2% soluble starch, 0.3% tryptone, 0.3% yeast extract and 0.1% K2HPO4 at 70 degrees C and pH 7.0, agitated at 200 rev min(-1) with 6-h-old inoculum (2% v/v) for 12 h. When the enzyme was partially purified using acetone (80%[v/v] saturation), a 43.7% recovery of enzyme with 6.2-fold purification was recorded. The KM and Vmax (soluble starch) values were 0.83 mg ml(-1) and 250 micromol mg(-1) protein min(-1), respectively. The enzyme was optimally active at 100 degrees C and pH 8.0 with a half-life of 3 h at 100 degrees C. Both alpha-amylase activity and production were Ca2+ independent. CONCLUSIONS: Bacillus thermooleovorans NP54 produced calcium-independent and thermostable alpha-amylase. SIGNIFICANCE AND IMPACT OF THE STUDY: The calcium-independent and thermostable alpha-amylase of B. thermooleovorans NP54 will be extremely useful in starch saccharification since the alpha-amylases used in the starch industry are calcium dependent. The use of this enzyme in starch hydrolysis eliminates the use of calcium in starch liquefaction and subsequent removal by ion exchange.

Bacillus↗
PubMed2001

Thermostable alpha-amylase production by an extreme thermophile Bacillus thermooleovorans.

AIMS: alpha-Amylase production by a newly isolated thermophile, Bacillus thermooleovorans, was studied under different cultivation conditions. METHODS AND RESULTS: The influence of various carbon and nitrogen sources on alpha-amylase production was quantified in batch fermentation in shake flasks. Starch and tryptone were observed to be the ideal carbon and nitrogen sources, respectively. Cultivation of the organism in a chemically defined medium consisting of glucose, riboflavin, cysteine, MgSO4, K2HPO4 and NaCl led to a near twofold increase in the production of alpha-amylase in comparison with that in the complex medium. The increase in enzyme production was achieved using vitamins and amino acids. When the organism was grown in a laboratory fermenter in the optimized complex medium, the noticeable effects were the near abolition of the lag phase, a 2.2-fold increase in enzyme production and a reduction in optimal production time from 12 to 4-5 h. CONCLUSION: Enhancement of amylase production was achieved under various cultivation conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacillus thermooleovorans produces a calcium-independent and thermostable amylase which can find use in starch saccharification.

Bacillus↗
PubMed2002

Characterization of a salt-tolerant extracellular a-amylase from Bacillus dipsosauri.

AIMS: The aims of this study were to purify and characterize an extracellular alpha-amylase from the salt-tolerant bacterium Bacillus dipsosauri. METHODS AND RESULTS: An extracellular alpha-amylase from B. dipsosauri strain DD1 was studied using the synthetic substrate 2-chloro-4-nitrophenyl-alpha-D-maltotrioside. Formation of the enzyme was induced by starch, repressed by D-glucose and highest after growth in medium containing 1.0 mol l-1 KCl. The alpha-amylase activity increased with KCl concentration, showed a pH optimum of 6.5, was stable up to 60 degrees C and was stimulated by 1.0 mol l-1 Na2SO4. The enzyme was purified from spent culture medium to apparent homogeneity by precipitation with ethanol, ion-exchange chromatography on DEAE-cellulose, centrifugal membrane filtration and gel-filtration chromatography on BioGel P-100. The purified enzyme had a denatured molecular mass of about 80 kDa but behaved on non-denaturing polyacrylamide gels as if it had a mass of about 30 kDa. The enzyme was partially inhibited by glucose-containing oligosaccharides of increasing length and strongly inhibited by the divalent cations Cd2+ and Zn2+. CONCLUSIONS: The extracellular alpha-amylase from B. dipsosauri strain DD1 was purified to homogeneity and found to exhibit an unusually high degree of salt tolerance. SIGNIFICANCE AND IMPACT OF THE STUDY: The alpha-amylase from B. dipsosauri differs from previously described enzymes and may be useful for the processing of starches under high-salt conditions.

Aerobiosis↗
PubMed2003

Production and partial characterization of a beta-amylase by Xanthophyllomyces dendrorhous.

AIMS: The characterization of a beta-amylase produced by Xanthophyllomyces dendrorhous. METHODS AND RESULTS: Growth in different culture media showed that X. dendrorhous produces an amylase whose synthesis is repressed by the carbon source and induced by starch and maltose. Enzymatic assays using substrates with different degrees of polymerization together with viscosity experiments revealed that the enzyme was beta-amylase. According to the biochemical characterization, the enzyme has a molecular weight of 240 kDa and a Km of 1.35 mg ml-1. The optimum pH and temperature were 5.5 and 50 degrees C, respectively. Using different inhibitors of the enzymatic activity it was shown that cysteine, tryptophan and serine are essential amino acids for catalysis. CONCLUSIONS: Xanthophyllomyces dendrorhous CECT1690 synthesizes and secretes beta-amylase that could be a by-product, in addition to carotenoid pigments, in the fermentation downstream. SIGNIFICANCE AND IMPACT OF THE STUDY: The beta-amylase produced by X. dendrorhous may have certain industrial applications.

Amino Acids↗
PubMedDate not supplied

[alpha-Amylase standard values in children].

In a group of 216 healthy children (age: 3 days-16 years) normal values of alpha-amylase in serum were found out (substrate: p-nitrophenyl-alpha, D-maltoheptaosid). Infants (age: 3-353 days) mostly showed lower catalytic concentrations than older children: alpha-amylase (25 degrees C) up to 30 U/L; alpha-amylase 37 degrees C): up to 60 U/L. But, the collective was too small (n = 30) to give any statistical statement, on the other hand biological and analytical deviations of catalytic concentrations were big. In children (n = 186; age: 1-16 years) the values were not dependent on sex or age. As normal range we propose for children aged 1-16 years alpha-amylase (25 degrees C): 20-110 U/L; alpha-amylase (37 degrees C): 40-200 U/L.

Adolescent↗
PubMed2000

Bean alpha-amylase inhibitor 1 in transgenic peas (Pisum sativum) provides complete protection from pea weevil (Bruchus pisorum) under field conditions.

Two alpha-amylase inhibitors, called alphaAI-1 and alphaAI-2, that share 78% amino acid sequence identity and have a differential specificity toward mammalian and insect alpha-amylases are present in different accessions of the common bean (Phaseolus vulgaris). Using greenhouse-grown transgenic peas (Pisum sativum), we have shown previously that expression of alphaAI-1 in pea seeds can provide complete protection against the pea weevil (Bruchus pisorum). Here, we report that alphaAI-1 also protects peas from the weevil under field conditions. The high degree of protection is explained by our finding that alphaAI-1 inhibits pea bruchid alpha-amylase by 80% over a broad pH range (pH 4.5-6.5). alphaAI-2, on the other hand, is a much less effective inhibitor of pea bruchid alpha-amylase, inhibiting the enzyme by only 40%, and only in the pH 4.0-4.5 range. Nevertheless, this inhibitor was still partially effective in protecting field-grown transgenic peas against pea weevils. The primary effect of alphaAI-2 appeared to be a delay in the maturation of the larvae. This contrasts with the effect of alphaAI-1, which results in larval mortality at the first or second instar. These results are discussed in relationship to the use of amylase inhibitors with different specificities to bring about protection of crops from their insect pests or to decrease insect pest populations below the economic injury level.

Animals↗
PubMed1992

A synthetic peptide of the rab3a effector domain stimulates amylase release from permeabilized pancreatic acini.

In this study we have employed a synthetic peptide of the rab3a effector domain, rab3AL, to examine whether a rab-like low molecular weight GTP-binding protein is involved in protein release from the rat pancreatic acinar cell. The peptide was found to be a potent stimulator of amylase release from streptolysin-O-permeabilized pancreatic acini, with an EC50 of approximately 60 microM. Stimulation of amylase discharge by rab3AL did not occur using either intact acini or permeabilized acini depleted of ATP. In contrast, a different effector domain peptide of the rab2 protein, rab2AL, a peptide with distinct sequence homology to rab3AL, was unable to stimulate amylase release, suggesting the specificity of the rab3AL response to rab3-like proteins. rab3AL stimulated release at [Ca2+] that were nonstimulatory in the absence of the peptide (10 nM). rab3AL potentiated the effect of guanosine 5'-[gamma-thio]triphosphate on amylase secretion and decreased the amount of guanosine 5'-[gamma-thio]triphosphate required for maximal secretion, suggesting that these two agents interact to modulate a distal step(s) of secretion. The above results provide functional evidence for the role of a rab-like low molecular weight GTP-binding protein and its effector protein(s) in the control of protein release from pancreatic acini. Because the discharge response to rab3AL is near the maximal obtainable from permeabilized acini, our results would suggest that rab3-like proteins control an important step in regulated secretion of amylase.

Amino Acid Sequence↗
PubMed1998

Characterization of the C-terminal propeptide involved in bacterial wall spanning of alpha-amylase from the psychrophile Alteromonas haloplanctis.

The antarctic psychrophile Alteromonas haloplanctis secretes a Ca2+- and Cl--dependent alpha-amylase. The nucleotide sequence of the amy gene and the amino acid sequences of the gene products indicate that the alpha-amylase precursor is a preproenzyme composed by the signal peptide (24 residues), the mature alpha-amylase (453 residues, 49 kDa), and a long C-terminal propeptide or secretion helper (192 residues, 21 kDa). In cultures of the wild-type strain, the 70-kDa precursor is secreted at the mid-exponential phase and is cleaved by a nonspecific protease into the mature enzyme and the propeptide. The purified C-terminal propeptide displays several features common to beta-pleated transmembrane proteins. It has no intramolecular chaperone function because active alpha-amylase is expressed by Escherichia coli in the absence of the propeptide coding region. In E. coli, the 70-kDa precursor is directed toward the supernatant. When the alpha-amylase coding region is excised from the gene, the secretion helper can still promote its own membrane spanning. It can also accept a foreign passenger, as shown by the extracellular routing of a beta-lactamase-propeptide fusion protein.

Amino Acid Sequence↗
PubMed2002

Rice alpha-amylase transcriptional enhancers direct multiple mode regulation of promoters in transgenic rice.

Expression of alpha-amylase genes in cereals is induced by both gibberellin (GA) and sugar starvation. In a transient expression assay, a 105-bp sugar response sequence (SRS) in the promoter of a sugar starvation highly inducible rice alpha-amylase gene, alphaAmy3, was shown previously to confer sugar response and to enhance the activity of the rice Act1 promoter in rice protoplasts. A 230-bp SRS-like sequence was also found in the promoter of another sugar starvation highly inducible rice alpha-amylase gene, alphaAmy8. The alphaAmy8 SRS contains a GA response sequence and was designated as alphaAmy8 SRS/GARS. In the present study, a transgenic approach was employed to characterize the function of the alpha-amylase gene SRSs in rice. We found that the alphaAmy3 SRS significantly enhances the endogenous expression pattern of the Act1 promoter in various rice tissues throughout their developmental stages. By contrast, the alphaAmy8 SRS/GARS significantly enhances Act1 promoter activity only in embryos and endosperms of germinating rice seeds. A minimal promoter fused to the alphaAmy8 SRS/GARS is specifically active in rice embryo and endosperm and is subject to sugar repression and GA induction in rice embryos. This sugar repression was found to override GA induction of alphaAmy8 SRS/GARS activity. Our study demonstrates that the alpha-amylase transcriptional enhancers contain cis-acting elements capable of enhancing endogenous expression patterns or activating sugar-sensitive, hormone-responsive, tissue-specific, and developmental stage-dependent expression of promoters in transgenic rice. These enhancers may facilitate the design of highly active and tightly regulated composite promoters for monocot transformation and gene expression. Our study also reveals the existence of cross-talk between the sugar and GA signaling pathways in cereals and provides a system for analyzing the underlying molecular mechanisms involved.

Base Sequence↗
PubMed1988

Comparison of gamma-glutamyl hydrolase (conjugase; EC 3.4.22.12) and amylase treatment procedures in the microbiological assay for food folates.

1. It has been suggested that deconjugation of food folates with pig kidney, compared with chicken pancreas, may increase measureable folate by approximately 50% (Phillips & Wright, 1983). Therefore deconjugation with conjugases from these two enzyme sources was optimized and compared. Folate was measured microbiologically with Lactobacillus casei (ATCC 7469) as the test organism at pH 5.6. 2. Treatment for 6 h with 200 mg pig kidney/200 mg sample was compared with the conventional assay employing overnight incubation with 20 mg chicken pancreas/5 g sample. Comparison of the deconjugation systems showed chicken pancreas to be superior for peas (Pisum sativum) and beans (Phaseolus vulgaris), while there was no difference for potatoes. 3. gamma-Glutamyl hydrolase (conjugase; EC 3.4.22.12) treatment for 2 and 20 h with pig kidney and chicken pancreas was optimized for raw potatoes and green frozen peas. Treatments with pig kidney were conducted at pH 4.6, which is optimal, and at pH 5.2. There was no significant difference between 2 and 20 h treatments at pH 5.2. Treatments with chicken pancreas were conducted at pH 6.1. Treatment for 2 h was preferred as it resulted in significantly higher measureable folate activity in peas and potatoes, and because overnight treatment can be influenced by microbial production of folate. 4. With optimal treatment conditions the source of enzymes did not significantly influence measureable folate activity. Chicken pancreas is the traditional source of conjugase in Scandinavia and was preferred for deconjugation. 5. Chicken pancreas, 20 and 60 mg, was used for deconjugation of sixteen different food samples, each containing approximately 200 ng folate. Chicken pancreas at 60 mg/sample gave significantly higher results. Further addition of enzyme did not increase the folate values. 6. A combined amylase treatment using heat-resistant alpha-amylase (EC 3.2.1.1) during extraction to ensure the gelatinization of the sample, and glucan 1,4-alpha-glucosidase (amyloglucosidase; EC 3.2.1.3), along with the incubation with chicken pancreas to complete the digestion, was shown to give a small but significant increase in folate values of starch-containing samples. 7. Folate results using the recommended procedure are given for raw potatoes, wheat bran, rolled oats, wheat flour and dark rye bread. 8. Chicken pancreas was shown to contain equally high amounts of amylases as did the alpha-amylase and amyloglucosidase sources. This finding may explain the relatively small benefit of a specific amylase addition.

Animals↗