Formation of Complex of Proteinous Animal α-Amylase Inhibitor (Haim) with Hog Pancreatic α-Amylase

A limited proteolysis of Avicel-adsorbable, Avicel-disintegrating endocellulase I (molecular weight 130,000) from Geotrichum candidum with subtilisin yielded a protein (molecular weight 80,000) which proved fully active toward soluble substrates such as CM-cellulose, but lost both the abilities to be adsorbed onto insoluble substrates and to disintegrate the cellulose fibres. An immunological experiment showed precipitin lines between endocellulase I and subtilisin-modified endocellulase in the pattern of partial identity. N-Bromosuccinimide-oxidized endocellulase I lost cellulase activity, but retained its adsorbability onto Avicel. It is suggested that endocellulase I had both the affinity site for adsorbing onto insoluble substrates and the ordinary active site.     

Isolation and Identification of a Hog Pancreatic α-Amylase Inhibitor (Haim) Producing Streptomyces

A screening test was carried out to obtain microbes which produce hog pancreatic α-amylase inhibitor and a new inhibitor was found in culture broth of an actinomycete, strain YM-25. This inhibitor was designated as Haim, an abbreviation for hog pancreatic α-amylase inhibitor from a microbe. The determined morphological and physiological properties of strain YM-25 led to the conclusion that the microorganism was Streptomyces griseosporeus.

When the microorganism was aerobically cultured at 30°C in a jar fermentor containing the most suitable medium for growth which consisted of 5% glycerol, 0.5% polypepton, 0.2% meat extract, 0.1% yeast extract, 0.4% Na₂HPO₄ ? 12H₂O, 0.1% KH₂PO₄, and 0.05% MgSO₄ ? 7H₂O (pH 7.3), the highest activity of Haim was obtained on 23~26hr cultivation.

Haim had specific inhibitory activities against animal α-amylases but not against microbial and plant α-amylases.     

Chemical Modification of Amino Groups of Acid-stable α-Amylase and Acid-unstable α-Amylase from Aspergillus niger

Monochlorotrifluoro-p-benzoquinone (CFQ) was used for investigating the state of the amino groups of acid-stable α-amylase and acid-unstable α-amylase. About half of the total amino groups in both enzyme molecules were reacted with the reagent. The unreactive amino groups seemed to exist in a different state from the reactive ones. Both enzymes whose amino groups were modified by CFQ still maintained the α-phenylmaltosidase activity in spite of losing or decreasing the amylase activity. These facts suggest that the amino groups of both enzymes were not in the active site but the modification of them caused steric hindrance.

The pH-stability of the acid-unstable α-amylase whose one or two amino groups were modified with succinic anhydride or 2,4,6-trinitrobenzene-l-sulfonate (TNBS) increased on the acidic side and decreased on the alkaline side, but further modification of them led to decrease the stability on both sides.     

Molecular Cloning and Expression of Proteinaceous α-Amylase Inhibitor Gene from Streptomyces nitrosporeus in Escherichia coli

The proteinaceous α-amylase inhibitor, T-76, gene was cloned by screening a Streptomyces nitrosporeus genomic library using a deoxyinosine-containing probe corresponding to the amino acid sequence of the inhibitor. The nucleotide sequence of the insert of a positive clone had an open reading frame of 330 bp that encoded a polypeptide of 110 amino acid residues with a calculated molecular mass of 11,306 daltons. The polypeptide begins with proximal basic amino acids and a region rich in hydrophobic amino acids that possibly act as a signal peptide for secretion, which is followed by a sequence consistent with the amino-terminal amino acid sequence of the T-76 inhibitor. Escherichia coli cells harboring the plasmid derivatives for expression produced the inhibitor in their periplasmic space. The amino-terminal sequence of the inhibitor produced by an E. coli transformant was identical to that of the T-76 inhibitor secreted by S. nitrosporeus. The amino acid sequence of the inhibitor deduced from nucleotide sequence showed significant homology to other proteinaceous α-amylase inhibitors.     

Purification of the Proteinaceous α-Amylase Inhibitor from Phaseolus Vulgaris by Affinity Chromatography with Immobilized Enzyme or Antibody

A protein inhibiting salivary and pancreatic a-amylase of mammalian origin is contained in dry seeds of beans (Phaseolus vulgaris). Starting from a crude bean extract, the amylase inhibitor may be purified about 30fold in one step to apparent homogeneity by chromatography on matrix-bound salivary amylase. Compared with protein obtained by a conventional purification procedure and in similar yield, the amylase inhibitor obtained by affinity chromatography had the same specific activity (4.5 (akat inhibitor units/mg protein). A one step purification from crude extracts to homogenous inhibitor with the same specific activity was achieved by immuno-affinity chromatography on immobilized rabbit antibody raised against pure amylase inhibitor. The yield was 60 % that of a conventional purification. Criteria of purity of the inhibitor protein were thin-layer electrofocussing and immuno-electrophoresis.     

Intestinal Absorbability of Wheat Allergens,Subunits of a Wheat α-Amylase Inhibitor,Expressed by Bacteria

Wheat CM2, CM3 and CM16 proteins are known as subunits of the tetrameric α-amylase inhibitor as well as major allergens to baker’s asthma. The purpose of this study is to produce these CM proteins by bacteria in a quantity adequate for studying thepenetration characteristics of the CM proteins through intestinal mcosa in rats and Caco-2 cells. cDNAs encoding the mature proteins were expressed in Escherichia coli and purified by an Ni²⁺-chelating column. The recombinant proteins were radioiodinated and admministrered orally to rats or applied to the apical site of the Caco-2 cell monolayer. The radioactivity in the trichloroacetic acid-insoluble fraction, which was mainly composed of peptides with molecular mass less than that of the intact CM proteins, in the serum and the basolateral medium was highest in recombinant CM3. Accordingly, the intestinal absorption of these three proteins in the form present in wheat should be evaluated.     

Chemical Modification of Sulfhydryl Groups in Porcine Pancreatic α-Amylase,and Purification and Properties of the Modified Amylase

The behavior of SH groups of porcine pancreatic α-amylase, called PPA II, was studied by chemical modification with 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB). Only two SH groups in PPA II reacted, in a pseudo-first-order reaction, and the modification was accompanied with the inactivation of the amylase. The reactivity of SH groups with DTNB was influenced by the ionic strength of the medium. The SH groups were protected against modification by the addition of some substrate analogs; maltopentaitol, maltotetraitol, maltotriitol and cyclomaltohexaose were effective analogs, whereas maltitol, d-glucitol and methyl α-d-glucoside did not protect these groups. The modified enzymes (M₁ and M₂), in which one and two SH groups reacted with DTNB, respectively, were purified in an electrophoretically homogeneous state by chromatography on Bio-Gel P-2 and TSK-Gel DEAE-Toyopearl 650S. The optimum pH of the modified enzyme (M₂) was 6.9~7.0, which was the same as that of the native PPA II. The isoelectric points of M₁ and M₂ were estimated to be 5.8 and 5.2, respectively, by the method of Catsimpoolas. The CD spectrum of PPA II was altered partially by the modification of SH groups with DTNB. Moreover, a precipitin line with a spur was observed in a double immunodiffusion test of PPA II and M₂ to rabbit antiserum of PPA II. It is concluded that the free SH group(s) in PPA II, located near the substrate binding site, don’t participate directly in its catalytic activity, but that the SH group(s) are involved in the antigenicity of PPA II.     

Purification and Primary Structure of Proteinous α-Amylase Inhibitor from Streptomyces chartreusis

A new polypeptide inhibitor, AI-409, that inhibits human salivary α-Amylase, was purified from a fermentation broth of Streptomyces chartreusis strain No. 409. This protein consists of a single-chain polypeptide of 78 amino acid residues, and includes two disulfide bridges. The primary structure of AI-409 and the locations of the disulfide bridges were identified by enzymatic digestion and the automatic Edman technique. Enzymatic digestion was done with trypsin, carboxypeptidase Y, and chymotrypsin. One of the disulfide bridges was between Cys(10) and Cys(26), and the other between Cys(44) and Cys(71).     

Properties and Specific Functional Features of Wheat Grain α-Amylase/Subtilisin Inhibitor

A protein bifunctional inhibitor of endogenous α-amylase and subtilisin has been isolated from wheat grain and purified. The inhibitor specifically inactivates α-amylase isozymes with high isoelectric point values (group α-AMY1) and has almost no effect on the α-AMY2 isozymes with low isoelectric point values. This enzyme does not belong to glycoproteins and has a molecular weight of 21 kDa and an isoelectric point of 7.2. The protein displays a relatively high thermostability and pH optimum of 8.0; its inhibitory activity requires the presence of Ca²⁺ cations. The inhibition of excess α-amylase in wheat grain with a low falling number by the purified protein is studied.     

Engineering of Barley α-Amylase

Abstract

Protein engineering of barley α-amylase addressed the roles of Ca²⁺ in activity and inhibition by barley α-amylase/subtilisin inhibitor (BASI), multiple attach in polysaccharide hydrolysis, secondary starch binding sites, and BASI hot spots in AMY2 recognition. AMY1/AMY2 isozyme chimeras faciliatated assignment of function to specific regions of the structure. An AMY1 fusion with starch binding domain and AMY1 mutants in the substrate binding cleft gave degree of multiple attack of 0.9–3.3, compared to 1.9 for wild-type. About 40% of the secondary attacks, succeeding the initial endo-attack, produced DP5-10 maltooligosaccharides in similar proportion for all enzyme variants, whereas shorter products, comprising about 25%, varied depending on the mutation. Secondary binding sites were important in both multiple attack and starch granule hydrolysis. Surface plasmon resonance and inhibition analyses indicated the importance of fully hydrated Ca²⁺ at the AMY2/BASI interface to strengthen the complex. Engineering of intermolecular contacts in BASI modulated the affinity for AMY2 and the target enzyme specificity.     

Isolation of a Raw Starch-Binding Fragment from Barley α-Amylase

Barley α-amylase was purified by ammonium sulfate fraction, ion-exchange, ultrafiltration, and gel filtration to homogeneity. The purified enzyme was partially digested with trypsin, and the reaction mixture was applied to a cyclohepta-amylose epoxy Sepharose 6B column. Bound fragments were eluted by free cyclohepta-amylose, lyophilized, and separated on Tricine gels. Four fragments were shown to interact with β-cyclodextrin. The fragment that could be identified on the gel with the lowest molecular weight (11 kDa) was electroblotted onto PVDF membrane for sequencing. The N-terminal sequence of this fragment was determined with the N-terminal amino acid corresponding to Ala283 in the whole protein. The trypsin cleavage was at Lys282/Ala283 and the C-terminal cleavage occurred at Lys354/Ile355 to give a fragment size of 11 kDa as estimated by SDS-PAGE. The fragment would be located at the C-terminal region, forming a majority of the antiparallel β-sheets in domain C and the α₇-and α₈-helices of the (α/β)₈ domain.     

Rice Bifunctional α-Amylase/Subtilisin Inhibitor: Cloning and Characterization of the Recombinant Inhibitor Expressed in Escherichia coli

The complete nucleotide sequences of the cDNA and its gene that encode a bifunctional α-amylase/subtilisin inhibitor of rice (Oryza sativa L.) (RASI) were analyzed. RASI cDNA (939 bp) encoded a 200-residue polypeptide with a molecular mass of 21,417 Da, including a signal peptide of 22 amino acids. Sequence comparison and phylogenetic analysis showed that RASI is closely related to α-amylase/subtilisin inhibitors from barley and wheat. RASI was found to be expressed only in seeds, suggesting that it has a seed-specific function. A coding region of RASI cDNA without the signal peptide was introduced into Escherichia coli and was expressed as a His-tagged protein. Recombinant RASI was purified to homogeneity in a single step by Ni-chelating affinity column chromatography and characterized to elucidate the target enzyme. The recombinant inhibitor had strong inhibitory activity toward subtilisin, with an equimolar relationship, comparable with that of native RASI, and weak inhibitory activity toward some microbial α-amylases, but not toward animal or insect α-amylases. These results suggest that RASI might function in the defense of the seed against microorganisms.     

Acid-Stable α-Amylase of Black Aspergilli

The acid-stable α-amylase or the acid-unstable α-amylase from Aspergillus niger contained 24 moles or 7 moles mannose and 4 moles or 1 mole hexosamine per mole of protein, respectively.

The acid-stable α-amylase and the acid-unstable α-amylase contained calcium only, but not detectable amounts of other metals. Calcium contents of the both enzymes were converged to at least one gram atom per mole of enzyme by dialysis against acetate buffer. The last calcium could be removed under the suitable conditions by EDTA. Calcium removal by EDTA was accompanied by the loss of activity and by the little change of UV absorption spectra. The phenomenon caused by calcium removal were partially reversible. This last one atom of calcium seemed to be essential for the maintenance of active structure of α-amylase.     

Acid-stable α-Amylase of Black Aspergilli

When black Aspergilli were cultivated in appropriate condition, culture filtrate showed dextrinizing activity even after acid treatment such as pH 2.5, at 37°C for 30 minutes. It suggested the existence of acid-stable dextrinizing amylase. To isolate this enzyme paper el-ectrophoretic procedure was carried out and the spot which showed acid-stable dextrinizing activity was obtained in addition to α-amylase and glucoamylase spots. This new amylase was purified by fractional precipitation with ammonium sulfate, rivanol and acetone, and was obtained in a crystalline form.     

Acid-stable α-Amylase of Black Aspergilli

The amino acid compositions of the acid-stable α-amylase and the acid-unstable α-amylase obtained from Aspergillus niger were determined by automatic column chromatography. The amino acid composition of the acid-unstable α-amylase was very similar to that of the α-amylase of Aspergillus oryzae. The amino acid composition of the acid-stable α-amylase was also similar in most part, but differed from that of the acid-unstable α-amylase in the following features, (a) The lysine content was lower, (b) Although the totals of carboxyl and amide were almost equal, there were considerably more free carboxyl residues, (c) The serine content was higher, (d) The proline content was lower. These facts may be related to the lower isoelectric point (pH 3.44) of the acid-stable α-amylase.

Amino-terminal amino acid analysis demonstrated one mole of amino-terminal leucine or isoleucine per mole of the acid-stable α-amylase and one mole of amino-terminal alanine per mole of the acid-unstable α-amylase.     

Acid-stable α-Amylase of Black Aspergilli

The Acid-stable α-amylase and the acid-unstable α-amylase from Aspergillus niger contained one mole of sulfhydryl group per one mole of enzyme, which probably existed correlating with calcium atom that was essential for the amylase activity.

Iodine reacted at acidic pH specifically with the sulfhydryl group of both enzymes and oxidized it to considerably high degree, since about 4 eq of iodine per mole of sulfhydryl group of both enzymes were consumed. The modification of the sulfhydryl group of the acid-stable α-amylase did not affect the amylase acitvity, while, that of the acid-unstable α-amylase reduced it to 70 per cents intact enzyme. It was difficult to carry out carboxy-methylation of the sulfhydryl group of the acid-stable α-amylase under mild conditions maintaining its activity, but that of the acid-unstable α-amylase was easily achieved.

These facts suggested that some differences existed in the neighborhood of the sulfhydryl group of both enzymes, and that the sulfhydryl group of them was not the active site.     

Acid-stable α-Amylase of Black Aspergilli

Some general properties of the acid-stable dextrinizing amylase of black Aspergillus were investigated comparing with those of Taka-amylase A. The mode of action on starch of this amylase was quite similar to that of Taka-amylase A. Saccharifying degree at red point in starch-iodine color reaction was 5.1% and the limit of starch saccharification was a little over 40 per cent calculated as glucose with both amylases. Maltase activity was absent. Degradation products in the course of starch hydrolysis were also quite similar and they mutarotated downward. So this amylase was decided to be α-type. Thermal stability of the acid-stable α-amylase was higher than that of Taka-amylase A. Its acid stability was much higher than that of Taka-amylase A. Taka-amylase A was inactivated completely at pH 2.2, 37°C, for 30 min, but the acid-stable α-amylase retained 87% of its original activity.

From the amylase preparation of black Aspergillus acid-stable α-amylase and acidunstable α-amylase were separated by gel filtration on sephadex G-100 column. From the acid-unstable α-amylase fraction this enzyme was purified by fractionations with rivanol and acetone, and finally obtained as a homogeneous protein after gel filtration with sephadex G-50. Comparison of some general properties between the two α-amylases was carried out. Catalytic action was quite similar with both enzymes, but dextrinizing unit per mg enzyme protein of the acid-unstable α-amylase was about 5.6 times as large as that of the acid-stable α-amylase. The acid-unstable α-amylase was less heat-stable than the acid-stable α-amylase. Acid stability and pH-activity curve were compared with both α-amylases. High stability of the acid-stable α-amylase in acidic condition was observed, but, in alkaline range, it was more sensitive than the acid-unstable α-amylase.     

Purification and Some Properties of an α-Amylase Inhibitor (Paim) from Streptomyces corchorushii

Paim, a microbial animal amylase inhibitor, was purified from the culture filtrate of Streptomyces corchorushii by salting out with ammonium sulfate and column chromatography on DEAE-cellulose, TEAE-cellulose, SP-Sephadex C-50, and Octyl Sepharose CL-4B. Paim was separated into 2 fractions (Paim I and II), both homogeneous on disc electrophoresis. The molecular weight of Paim I was 4100 and that of II, 4400 by amino acid analysis. Paim I and II consisted of 39 and 42 amino acid residues, respectively, and contained no lysine, isolecucine, or phenylalanine. Paim contained no carbohydrate moiety, and was stable even after being treated at 100°C for lOmin in the pH range from 5 to 8.