Production of α-amylase fromHalobacterium halobium

Maximum production of extracellular -amylase activity inHalobacterium halobium was at 40°C in a medium containing 25% (w/v) NaCl, 1% (w/v) soluble starch and 1% (w/v) peptone, in presence of 0.1mm ZnSO₄ after 5 days in shaking cultures. The amylase had optimal activity at pH 6.5 in the presence of 1 to 3% (w/v) NaCl at 53°C.S. Patel, N. Jain and D. Madamwar are with the Post Graduate Department of Biosciences, Sadar Patel University, Vallabh Vidyanagar-388120, India.     

Wheat protein inhibitors of α-amylase

The location in the seed, molecular properties and biological role of protein α-amylase inhibitors from wheat are discussed. Inhibition specificity of albumin inhibitors and structural features essential for interaction with inhibited amylases are also examined. The possible significance of these naturally occurring inhibitors in relation to their presence in foods in active form is described. Finally, genetic aspects of the albumin inhibitor production and the possibility of improving nutritional value and insect re     

Multiple forms of porcine pancreatic α-amylase

Porcine pancreatic α-amylase can be fractionated into two components by DEAE-cellulose chromatography and by disc electrophoresis. The basis for fractionation is tentatively ascribed to a charge difference. The two components displayed the same specific activity and their thermal and pH stability, as well as the variation of V_max and K_m with pH, were identical within experimental error. It is concluded that the multiple forms of the amylase are physically distinct, but structurally related, with a common active site.     

Mechanisms of action of the α-amylase of Micromonospora melanosporea

The -amylase of Micromonospora melanosporea was produced extracellularly during batch fermentation in a 5.0-1 fermentor. The absence of an organic nitrogen source in its growth medium facilitated subsequent purification of the enzyme by ammonium sulphate fractionation and two consecutive Superose-12 gel-filtration steps. The enzyme exhibited maxima for activity at pH 7.0 and 55° C and was 72% stable at pH 6.0–12.0 for 30 min at 40° C. It had a relative molecular mass of 45 000 and an isoelectric point at pH 7.6. The enzyme catalyses the conversion of starch to maltose (53%, w/w) as the predominant final end-product. Initial hydrolysis of this substrate, however, gave rise to the formation of maltooligosaccharides in the range maltotriose to maltohexaose. Maximum yields of these intermediate sugars accumulated to between 31 and 42% (w/w) as the reaction proceeded. The action of the M. melanosporea amylase on high concentrations of saccharides larger than maltotriose resulted in the formation of mainly maltose and maltotriose without concomitant glucose production. A combination of hydrolytic and transfer events is postulated to be responsible for this phenomenon and for the high maltose levels achieved. Correspondence to: C. T. Kelly     

Purification of α-amylase from Bacillus lentus cultures

Acetone fractionation of Bacillus lentus culture filtrate yielded the highest -amylase activity and the 66.6% fraction reached 13-fold that of the crude enzyme preparation. Gel filtration and ion exchange chromatography afforded a pure -amylase (relative molecular mass, 42 000). The pure enzyme was highly active on starch and dextrin. It produced a mixture of oligosaccharides as major products of starch hydrolysis. Maximal activity was reached at 70° C and pH 6.1. Ca²⁺, Na⁺, K⁺ and Sr²⁺ ions stabilized or slightly stimulated the enzyme whereas Ag⁺, Co²⁺, Hg²⁺, Zn²⁺, Cd²⁺ and Fe³⁺ ions strongly inhibited the activity. The enzyme contained 16 amino acids, of which aspartic and glutamic acids were present in the highest proportions. Correspondence to: S. H. Omar     

The high maltose-producing α-amylase of Penicillium expansum

Summary An -amylase capable of producing exceptionally high levels of maltose (74%) from starch has been identified from a strain of Penicillium expansum. The enzyme is produced extracellularly and was purified to homogeneity by starch adsorption and Sephadex gel filtration chromatography. P. expansum -amylase has a pH optimum of 4.5 and is stable in the pH range of 3.6–6.0. Other properties include a temperature optimum of 60° C, a molecular weight of 69 000 and an isoelectric point of 3.9. The most outstanding feature of the P. expansum enzyme is its ability to yield 14% more maltose and 17.1% less maltotriose than a currently used commercial enzyme. This may be partly explained by the greater affinity of this new enzyme for maltotriose (K _m=0.76 mM) relative to the commerical enzyme, Fungamyl (K _m=2.9 mM). The enzyme reported here is unique among fungal -amylases in being able to produce such high levels of maltose and its physicochemical properties suggest that it has potential for commercial development.     

Stability of fungal α-amylase in sodium dodecylsulfate

Unfolding of a fungal -amylase in aqueous sodium dodecylsulfate (SDS) solution was examined by SDS-polyacrylamide gel electrophoresis (PAGE). When the -amylase was incubated with 1% SDS at room temperature and subjected to SDS-PAGE, it showed a much higher mobility than expected from the molecular weight. Circular dichroic and gel filtration analyses indicated that the protein is apparently in the native conformation upon incubation with 1% SDS. When the protein was heated in the presence of 1% SDS at 90°C for 10 min, it had a lower mobility in SDS-PAGE and showed characteristics of an unfolded protein by circular dichroism and gel filtration. The melting temperatures of the protein were determined in the absence and presence of SDS by incubating it for 10 min at various temperatures. The melting temperatures were 70, 55, and 49°C in the presence of 0, 1, and 2% SDS, respectively. The observed small shift of the melting temperatures by SDS suggests that the destabilizing action of SDS on the -amylase is weak. However, the unfolding in SDS is not reversible process, since prolonged incubation of the protein with 1% SDS at 50°C gradually increased the amount of unfolded protein. This indicates that the SDS-induced unfolding of the -amylase is a slow process.     

Effects of bean and wheat α-amylase inhibitors on α-amylase activity and growth of stored product insect pests

Insect α-amylase inhibiting and/or growth inhibiting activities of proteinaceous inhibitors from red kidney bean (Phaseolus vulgaris) and hard red winter wheat (Triticum aestivum) were examined. The bean inhibitor was most effectivein vitro against α-amylases from the red flour beetle (Tribolium castaneum) and the confused flour beetle (T. confusum), followed by those from the rice weevil (Sitophilus oryzae) and yellow mealworm (Tenebrio molitor). The insect enzymes were from two- to 50-fold more susceptible than human salivary α-amylase. When the inhibitors were added at a 1% level to a wheat flour plus germ diet, the growth of red flour beetle larvae was slowed relative to that of the control group of larvae, with the bean inhibitor being more effective than the wheat inhibitor. Development of both the red flour beetle and flat grain beetle (Cryptolestes pusillus) was delayed by 1% bean inhibitor, but development of the sawtoothed grain beetle (Oryzaephilus surinamensis) and lesser grain borer (Rhyzopertha dominica) was not affected by either the bean or wheat inhibitor at the 1% level. Rice weevil adults fed a diet containing 1% bean or wheat inhibitor exhibited more mortality than weevils fed the control diet. When the wheat amylase inhibitor was combined with a cysteine protease inhibitor, E-64, and fed to red flour beetle larvae, a reduction in the growth rate and an increase in the time required for adult eclosion occurred relative to larvae fed either of the inhibitors separately. The bean inhibitor was just as effective alone as when it was combined with the protease inhibitor. These results demonstrate that plant inhibitors of insect digestive enzymes act as growth inhibitors of insects and possibly as plant defense proteins, and open the way to the use of the genes of these inhibitors for genetically improving the resistance of cereals to storage pests. Cooperative investigation between the Agricultural Research Service, the University of California, San Diego, and the Kansas Agricultural Experiment Station (Contribution no. 94-416-J). Supported in part by a grant from the Ministry of Education and Science, Spain-Fulbright Program to J.J.P. Mention of a proprietary product does not constitute a recommendation or endorsement by the USDA. The USDA is an equal opportunity/affirmative action employer and all agency services are available without discrimination.     

Purification and characterization of α-amylase fromAspergillus flavus

Aspergillus flavus produced approximately 50 U/mL of amylolytic activity when grown in liquid medium with raw low-grade tapioca starch as substrate. Electrophoretic analysis of the culture filtrate showed the presence of only one amylolytic enzyme, identified as an α-amylase as evidenced by (i) rapid loss of color in iodine-stained starch and (ii) production of a mixture of glucose, maltose, maltotriose and maltotetraose as starch digestion products. The enzyme was purified by ammonium sulfate precipitation and ion-exchange chromatography and was found to be homogeneous on sodium dodecyl sulfate— polyacrylamide gel electrophoresis. The purified enzyme had a molar mass of 52.5±2.5 kDa with an isoelectric point at pH 3.5. The enzyme was found to have maximum activity at pH 6.0 and was stable in a pH range from 5.0 to 8.5. The optimum temperature for the enzyme was 55°C and it was stable for 1 h up to 50°C. TheK _m andV for gelatinized tapioca starch were 0.5 g/L and 108.67 μmol reducing sugars per mg protein per min, respectively.     

Isoenzymes of α-amylase during pod development of field beans

The pod mesophyll of field beans accumulates large amounts of starch during stage 1 of embryogenesis, which is later utilized during stage 2. The activity of starch degradation in the pod is under metabolic control of the enclosed seeds. Changes in the isoenzyme pattern of α-amylase and not starch phosphorylase coincide with the beginning of the starch degradation period in the pods. Mesophyll cells of the pods contain the same α-amylase isoenzymes as the endocarp but exhibit a higher α-amylase activity that parallels the much higher starch content of this tissue in comparison to the endocarp. Regulation of starch breakdown may be mediated at least in part by the formation of a special α-amylase isoenzyme.     

Biochemical genetics of α-amylase isozymes of the chicken pancreas

In the chicken population at large, three electrophoretically distinct pancreatic alpha-amylase isozymes were discovered. The isozymes were designated Pa 1, Pa 2, and Pa 3. The local population of chickens, however, possessed only isozymes Pa 2 and Pa 3 present as three phenotypes: Amy-2 B, consisting of isozyme Pa2; Amy2 BC, consisting of isozymes Pa 2 plus Pa 3; and Amy2 C, consisting of isozyme Pa 3. Pancreatic biopsy permitted the establishment of a breeding flock with defined amylase phenotypes. Matings of this flock established that amylases are inherited as codominant alleles at a single genetic locus. Further, there was no evidence of ontogenetic modification of the amylase isozymes. It was observed that amylase isozymes Pa 2 and Pa 3 each generated a family of at least three faster-migrating amylolytic proteins. These post-translationally modified amylases were designated Pa Xa, Pa Xb, and Pa Xc, where X represents the number of the progenitor amylase. Structural analyses of purified amylases demonstrated that all amylase isozymes are nonglycosidated, monomeric molecules of molecular weight 55,000. In addition, the data are consistent with the hypothesis that the faster-migrating amylases are produced by deamidation of asparagine and/or glutamine residues.     

Expression of α-amylase in cultured callus of french bean

α-Amylase (EC 3.2.1.1) expression was found in calli of French bean (Phaseolus vulgaris L. cv Goldstar). We examined enzyme activity in the calli to investigate influence of gibberellin and sugars on enzyme expression. After subculture of the calli, α-amylase activity decreased, and then increased at a stationary phase of callus growth. Exogenous application of gibberellin and an inhibitor of gibberellin synthesis, uniconazole, did not have any significant effects on the enzyme expression. Sugar starvation increased the activity, while addition of metabolizable sugars, such as sucrose, glucose and maltose, to the medium repressed expression. Addition of 6% mannitol, a non-metabolizable sugar, to the medium induced higher α-amylase expression as compared to addition of 3% mannitol. This result suggests that osmotic stress enhances α-amylase activity in the calli. Furthermore, high concentrations of agar in the medium increased α-amylase activity in the calli. It is probable that high concentrations of agar prevented incorporation of nutrient into the calli and induced the α-amylase activity in the calli.     

Comparison of wheat albumin inhibitors of α-amylase and trypsin

Wheat albumins were extracted from whole wheat flour with 150 mM sodium chloride solution and precipitated between 0·4 and 1·8 M ammonium sulphate. The albumin precipitate was separated by gel filtration on Sephadex G100 into five peaks. Three peaks (II, III, and IV), whose MWs were 60 000, 24 000 and 12 500 daltons respectively, were active toward several insect α-amylases, whereas only peak III inhibited human saliva and pancreatic α-amylases. Peaks III and IV also inhibited trypsin. In each active peak, we found several α-amylase inhibitors slightly different in their electrophoretic mobilities in a Tris—glycine buffer system (pH 8·5), whereas only one major trypsin inhibitor was present in peaks III and IV. In contrast to α-amylase inhibitors that were all anodic, trypsin inhibitors migrated to the cathode under our experimental conditions. From a quantitative standpoint, wheat albumins that inhibit trypsin are negligible, whereas about 2/3 of the total albumin inhibits amylases from different origins. All inhibitor components of peak III were active toward both insect and mammalian α-amylases. Moreover, they reversibly dissociated in the presence of 6 M guanidine hydrochloride giving two similar subunits.     

Molecular characterization of a bean α-amylase inhibitor that inhibits the α-amylase of the Mexican bean weevil Zabrotes subfasciatus

Cultivated varieties of the common bean (Phaseolus vulgaris L.) contain an α-amylase inhibitor (αAI-1) that inhibits porcine pancreatic α-amylase (PPA; EC 3.2.1.1) and the amylases of certain seed weevils, but not that of the Mexican bean weevil, Zabrotes subfasciatus. A variant of αAI-1, called αAI-2, is found in certain arcelin-containing wild accessions of the common bean. The variant αAI-2 inhibits Z. subfasciatus α-amylase (ZSA), but not PPA. We purified αAI-2 and studied its interaction with ZSA. The formation of the αAI-2-ZSA complex is time-dependent and occurs maximally at pH 5.0 or below. When a previously isolated cDNA assumed to encode αAI-2 was expressed in transgenic tobacco seeds, the seeds contained inhibitory activity toward ZSA but not toward PPA, confirming that the cDNA encodes αAI-2. The inhibitors αAI-1 and αAI-2 share 78% sequence identity at the amino acid level and they differ in an important region that is part of the site where the enzyme binds the inhibitor. The swap of a tripeptide in this region was not sufficient to change the specificity of the two inhibitors towards their respective enzymes. The three-dimensional structure of the αAI-1/PPA complex has just been solved and we recently obtained the derived amino acid sequence of ZSA. This additional information allows us to discuss the results described here in the framework of the amino acid residues of both proteins involved in the formation of the enzyme-inhibitor complex and to pinpoint the amino acids responsible for the specificity of the interaction. Received: 14 April 1997 / Accepted: 10 May 1997     

Enhancement of α-amylase production by integrating and amplifying the α-amylase gene of Bacillus amyloliquefaciens in the genome of Bacillus subtilis

Summary The -amylase gene of Bacillus amyloliquefaciens was integrated into the genome of Bacillus subtilis by homologous recombination. In the first transformation step, several strains were obtained carrying the -amylase gene as two randomly located copies. These strains produced -amylase in the quantities comparable with that of the multicopy plasmid pKTH10, carrying the same -amylase gene. With the plasmid system, however, the rate of the -amylase synthesis was faster and the production phase shorter than those of the chromosomally encoded -amylase. The two chromosomal gene copies were further multiplied either by amplification using increasing antibiotic concentration as the selective pressure or by performing a second transformation step, identical to the first integration procedure. Both methods resulted in integration strains carrying up to eight -amylase gene copies per one genome and producing up to eightfold higher -amylase activity than the parental strains. Six out of seven transformants, studied in more detail, were stable after growth of 42 h even without antibiotic selection. The number of the DNA and mRNA copies of the -amylase gene was quantitavely determined by sandwich hybridization techniques, directly from culture medium.     

Inhibitors of α-glucosidase, α-amylase and lipase from Chrysanthemum morifolium

A new endoperoxysesquiterpene lactone, 10α-hydroxy-1α,4α-endoperoxy-guaia-2-en-12,6α-olide (1), together with a flavanone, eriodictyol (2), and two flavone glycosides, acacetin-7-O-β-d-glucopyranoside (3) and acacetin-7-O-α-l-rhamopyranoside (4), were isolated from the methanol extract of Chrysanthemum morifolium flowers by a bioassay-guided fractionation. Compound 1 showed strong inhibitory effects against α-glucosidase and lipase activities, with IC₅₀ values of 229.3 and 161.0 μM, respectively. The flavone glycosides 3 and 4 inhibited both α-glucosidase and α-amylase, while flavanone 2 was only effective against α-amylase.     

Action pattern of human pancreatic α-amylase on maltoheptaose,a substrate for determining α-amylase in serum

An enzymatic assay for the determination of α-amylase in serum was developed which employed a soluble substrate, maltoheptaose, and a coupled enzymatic indicator reaction consisting of α-glucosidase and the hexokinase—glucose-6-phosphate dehydrogenase system. We used high-performance liquid chromatography (HPLC) to establish the action pattern of maltoheptaose under the test conditions: (A) the action pattern of α-amylase alone, (B) that of the combined action of α-amylase and α-glucosidase. Conducive to this effort was: the availability of pure maltoheptaose and human pancreatic α-amylase; the development of an adequate procedure for sample pretreatment (partition chromatography on a mixed-bed ion exchanger) and of an HPLC system for separation of substrate and reaction products without interference from by-products of the assay (partition chromatography on a cation-exchange column with acetonitrile—water); and the use of a new, very sensitive refractometric detector revealing sugar amounts as low as 40 ng.We derived the following stoichiometric equations:

The standard deviation of the rate coefficients is about 5%.     

The α-amylase of the beetle Callosobruchus chinensis properties

C. chinensis larval amylase is activated by Ca(2+) and inhibited by Cl(-) and EDTA (K(i) 6.7x10(-3)m). GSH and 2-mercaptoethanol activate, presumably at different sites, as 2-mercaptoethanol interferes with Ca(2+) activation, whereas GSH enhances it. The inhibition by iodoacetic acid and N-ethylmaleimide (K(i) 1.55x10(-2)m) suggest that free thiol groups are essential for activity. The pH optimum of 5.2-5.4 is moved to 5.6-5.8 by Ca(2+) and 2-mercaptoethanol. The activation energy is 7270 cal/mol, and is not affected by Ca(2+) and 2-mercaptoethanol. K(m) for soluble starch is 2.3mg/ml.     

Hydrolysis of starch particles using immobilized barley α-amylase

Barley α-amylase has been immobilized on silica particles with diameters between 0.5 and 10 μm using a covalent binding method. Immobilization procedures were adjusted to optimize enzyme activity. The effects of product inhibition, thermal stability and operational stability have been determined. The feasibility of using the immobilized enzyme to hydrolyze wheat starch particles at temperatures below the gelatinization temperature (<55 °C) was proven. The optimal conditions for the hydrolysis were found to be: pH 4.5, 40 °C, calcium ion concentration 0.002 M and immobilized enzyme loading of 30 mg/ml. At these conditions, the immobilized enzyme was able to hydrolyze wheat starch particles at concentrations as high as 100 mg/ml with a final conversion of 90% after 24 h of operation. Maltose and glucose were found to inhibit the immobilized enzyme in a similar manner as reported previously using soluble enzyme. Although the thermostability of the immobilized enzyme was superior to the soluble enzyme, the immobilized enzyme degraded at the same rate as the soluble enzyme during cold wheat starch hydrolysis (operational stability unchanged). Model equations are presented for product inhibition, hydrolysis kinetics and enzyme degradation. Using best-fit parameters, the equations are shown to fit the experimental data well.