Enzymatic degradation of granular potato starch by <Emphasis Type="Italic">Microbacterium aurum</Emphasis> strain B8.A

Microbacterium aurum strain B8.A was isolated from the sludge of a potato starch-processing factory on the basis of its ability to use granular starch as carbon- and energy source. Extracellular enzymes hydrolyzing granular starch were detected in the growth medium of M. aurum B8.A, while the type strain M. aurum DSMZ 8600 produced very little amylase activity, and hence was unable to degrade granular starch. The strain B8.A extracellular enzyme fraction degraded wheat, tapioca and potato starch at 37 °C, well below the gelatinization temperature of these starches. Starch granules of potato were hydrolyzed more slowly than of wheat and tapioca, probably due to structural differences and/or surface area effects. Partial hydrolysis of starch granules by extracellular enzymes of strain B8.A resulted in large holes of irregular sizes in case of wheat and tapioca and many smaller pores of relatively homogeneous size in case of potato. The strain B8.A extracellular amylolytic system produced mainly maltotriose and maltose from both granular and soluble starch substrates; also, larger maltooligosaccharides were formed after growth of strain B8.A in rich medium. Zymogram analysis confirmed that a different set of amylolytic enzymes was present depending on the growth conditions of M. aurum B8.A. Some of these enzymes could be partly purified by binding to starch granules.     

The high maltose-forming α-amylase ofSaccharomonospora viridis: mechanisms of action

Summary The thermophilic actinomycete,Saccharomonospora viridis produces a thermostable -amylase which forms 63% (w/w) maltose on hydrolysis of starch. Maltotriose and maltotetraose are the only intermediate products observed during this reaction, with maltotriose accumulating to 40% (w/w). Both unimolecular and multimolecular mechanisms (transfers and condensation) have been shown to occur during the concentration-dependent degradation of maltotriose and maltotetraose. Such reactions result in the almost exclusive formation of maltose from maltotriose at high initial concentration. These mechanisms of action result in the production of the high levels of maltose obtained upon hydrolysis of starch and related substrates.     

The raw starch-degrading alkaline amylase ofBacillus sp IMD 370

The amylase ofBacillus sp IMD 370 is the first report of an alkaline amylase with the ability to digest raw starch. The amylase could degrade raw corn and rice starches more effectively than raw potato starch. It showed no adsorb-ability to any type of raw starch at any pH value tested. The enzyme digested raw corn starch to glucose, maltose, maltotriose and maltotetraose. The maximum pH for raw starch hydrolysis was pH 8.0 compared to pH 10.0 for soluble starch hydrolysis. The metal chelator, ethylenediaminetetraacetic acid, strongly inhibited raw starch-digestion and its effect was reversed by the addition of divalent cations. Degradation of raw starch was stimulated six-fold in the presence of -cyclodextrin (17.5 mM).     

Production of native-starch-degrading enzymes by a Bacillus firmus/lentus strain

Summary A bacterium belonging to the Bacillus firmus/lentus-complex and capable of growth on native potato starch was isolated from sludge of a pilot plant unit for potato-starch production. Utilization of a crude enzyme preparation obtained from the culture fluid after growth of the microorganism on native starch, resulted in complete degradation of native starch granules from potato, maize and wheat at a temperature of 37°C. Glucose was found as a major product. Production of maltose, maltotriose and maltotetraose was also observed. Native-starch-degrading activity (NSDA) could be selectively adsorbed on potato-starch granules, whereas soluble-starch-degrading activity (SSDA) remained mainly in solution. The use of such a starch-adsorbed enzyme preparation on native starch resulted in a completely changed product pattern. An increase in oligosaccharides concomitant with less glucose formation was observed. An increased conversion of soluble starch to maltopentaose was possible with this starch-adsorbed enzyme preparation. It is concluded that NSDA comes from -amylase(s) and SSDA from glucoamylase(s) and/or -glucosidase(s). Cultivation of B. firmus/lentus on glucose, maltose, or soluble starch resulted in substantially smaller quantities of (native) starch-degrading activity.Offprint requests to: D. J. Wijbenga     

Organisation of the external region of the starch granule as determined by infrared spectroscopy

Attenuated total reflectance-Fourier transform infrared (ATR-FTIR) was used to study the external regions of starch granules. Native starches (wheat, potato, maize, waxy maize and amylomaize) were analysed and compared to gelatinised and acid-hydrolysed starches. The IR spectra of potato and amylomaize starches were closer to that of highly ordered acid-hydrolysed starch than the other starches. FTIR was not able to differentiate between A- and B-type crystallinity so the difference observed between starches was not related to this factor. The variation between starch varieties was interpreted in terms of the level of ordered structure present on the edge of starch granules with potato and amylomaize being more ordered on their outer regions. This could explain the high resistance of both these starches to enzyme hydrolysis.     

Production and Characteristics of Raw-Potato-Starch-Digesting alpha-Amylase from Bacillus subtilis 65

A newly isolated bacterium, identified as Bacillus subtilis 65, was found to produce raw-starch-digesting alpha-amylase. The electrophoretically homogeneous preparation of enzyme (molecular weight, 68,000) digested and solubilized raw corn starch to glucose and maltose with small amounts of maltooligosaccharides ranging from maltotriose to maltoheptaose. This enzyme was different from other amylases and could digest raw potato starch almost as fast as it could corn starch, but it showed no adsorbability onto any kind of raw starch at any pH. The mixed preparation with Endomycopsis glucoamylase synergistically digested raw potato starch to glucose at 30 degrees C. The raw-potato-starch-digesting alpha-amylase showed strong digestibility to small substrates, which hydrolyzed maltotriose to maltose and glucose, and hydrolyzed p-nitrophenyl maltoside to p-nitrophenol and maltose, which is different from the capability of bacterial liquefying alpha-amylase.     

Molecular cloning, nucleotide sequencing, and expression of the Bacillus subtilis (natto) IAM1212 alpha-amylase gene, which encodes an alpha-amylase structurally similar to but enzymatically distinct from that of B. subtilis 2633.

An alpha-amylase gene of Bacillus subtilis (natto) IAM1212 was cloned in a lambda EMBL3 bacteriophage vector, and the nucleotide sequence was determined. An open reading frame encoding the alpha-amylase (AMY1212) consists of 1,431 base pairs and contains 477 amino acid residues, which is the same in size as the alpha-amylase (AMY2633) of B. subtilis 2633, an alpha-amylase-hyperproducing strain, and smaller than that of B. subtilis 168, Marburg strain. The amino acid sequence of AMY1212 is different from that of AMY2633 at five residues. Enzymatic properties of these two alpha-amylases were examined by introducing the cloned genes into an alpha-amylase-deficient strain, B. subtilis M15. It was revealed that products of soluble starch hydrolyzed by AMY1212 are maltose and maltotriose, while those of AMY2633 are glucose and maltose. From the detailed analyses with oligosaccharides as substrates, it was concluded that the difference in hydrolysis products of the two similar alpha-amylases should be ascribed to the different activity hydrolyzing low-molecular-weight substrates, especially maltotriose; AMY1212 slowly hydrolyzes maltotetraose and cannot hydrolyze maltotriose, while AMY2633 efficiently hydrolyzes maltotetraose and maltotriose. Further analyses with chimeric alpha-amylase molecules constructed from the cloned genes revealed that only one amino acid substitution is responsible for the differences in hydrolysis products.     

Interaction between dry starch and plasticisers glycerol or ethylene glycol, measured by differential scanning calorimetry and solid state NMR spectroscopy

The interaction of crystalline amylose and of crystalline and amorphous amylopectin with the plasticisers glycerol or ethylene glycol in the absence of water was studied, by using differential scanning calorimetry (DSC) and solid state nuclear magnetic resonance (NMR) spectroscopy. Upon heating starch freshly mixed with plasticisers, a strong exothermal interaction enthalpy of ΔH−35 J/g was detected by DSC. At room temperature glycerol interacts mainly with the amorphous starch regions, the interaction taking 8 days to reach equilibrium. For ethylene glycol the interaction is faster, taking four days to reach equilibrium, and the rate is not affected by crystallinity. Ethylene glycol interacts in a more ordered manner with amorphous than with crystalline material, resulting in a narrower ethylene glycol cross-polarisation magic angle spinning (CP/MAS) signal when equilibrium is reached at room temperature. Upon heating, more glycerol or ethylene glycol is immobilised, but in a less ordered manner than upon storage at room temperature. This results in a more intense, but broader plasticiser CP/MAS signal upon heating. Interaction in a more ordered manner probably implies interaction with more of the hydroxy groups of the plasticiser. The polysaccharide mobility is increased more when the plasticiser interacts in a more ordered manner, as observed by small starch signals in HP/DEC spectra.     

Purification and characterization of a thermostable raw starch digesting amylase from a Streptomyces sp. isolated in a milling factory

A raw starch utilizing microbe was isolated from mud in a milling factory. The 16S ribosomal DNA (rDNA) sequencing and morphological properties of the strain indicated that it belongs to the genus Streptomyces. A strongly raw starch digesting amylase was purified from the culture supernatant of the strain by chromatographic procedures. The specific activity of the enzyme was 11.7 U/mg, molecular mass 47 kDa, optimum pH 6.0, and optimum temperature 50 to 60 degrees C. The enzyme showed sufficient activity even at 70 degrees C. It was activated by calcium, cobaltous, and magnesium ions, and inhibited by copper, nickel, zinc, and ferrous ions. It formed maltose mainly from raw and gelatinized starch, and glycogen. No products were formed from glucose, maltose, maltotriose, pullulan, or cyclodextrins (CDs). The enzyme digested raw wheat, rice, and waxy rice starch rapidly, and raw corn, waxy corn, sweet potato, tapioca, and potato starch normally.     

Hydrolysis of aryl beta-maltotriosides by sweet potato beta-amylase and soybean beta-amylase.

Sweet potato beta-amylase [EC 3.2.1.2, alpha 1,4-D-glucan maltohydrolase]-catalyzed hydrolyses of aryl beta-maltotriosides with substituents, NO2-, Cl-, and Br- at the o-, m-, and p-positions in the phenyl ring were studied at pH 4.8 and 25 degrees C. The hydrolyses of a few of the maltotriosides by soybean beta-amylase [EC 3.2.1.2, alpha-1,4-D-glucan maltohydrolase] were also studied at pH 5.4 and 25 degrees C. It was found that the aryl beta-maltotriosides were preferentially hydrolyzed into maltose and aryl beta-D-glucosides by both beta-amylases. The Michaelis constant Km and the molecular activity ko were determined for the hydrolyses of these maltotriosides and compared with those of maltotriose and maltotetraose. Aryl beta-maltotriosides were more rapidly hydrolyzed than maltotriose by a factor of 30--80, and more slowly hydrolyzed than maltotetraose by a factor of 10--30, depending on the kinds of substituents. The rapid hydrolysis of aryl beta-maltotrioside as compared with maltotriose may be due to the interaction of an aryl group with the subsite of beta-amylase. This is in contrast with glucoamylase [EC 3.2.1.3, alpha-1,4-D-glucan glucohydrolase] of Rhizopus niveus-catalyzed hydrolysis of phenyl beta-maltoside, whose phenyl group does not interact so much with the subsite of the enzyme.     

Production and Characteristics of Raw-Potato-Starch-Digesting α-Amylase from Bacillus subtilis 65

A newly isolated bacterium, identified as Bacillus subtilis 65, was found to produce raw-starch-digesting α-amylase. The electrophoretically homogeneous preparation of enzyme (molecular weight, 68,000) digested and solubilized raw corn starch to glucose and maltose with small amounts of maltooligosaccharides ranging from maltotriose to maltoheptaose. This enzyme was different from other amylases and could digest raw potato starch almost as fast as it could corn starch, but it showed no adsorbability onto any kind of raw starch at any pH. The mixed preparation with Endomycopsis glucoamylase synergistically digested raw potato starch to glucose at 30°C. The raw-potato-starch-digesting α-amylase showed strong digestibility to small substrates, which hydrolyzed maltotriose to maltose and glucose, and hydrolyzed p-nitrophenyl maltoside to p-nitrophenol and maltose, which is different from the capability of bacterial liquefying α-amylase.     

Purification and some properties of a starch debranching enzyme ofHendersonula toruloidea

An extracellular, debranching isoamylase fromHendersonula toruloidea ATCC 64930, grown on starch, was purified 12-fold to an electrophoretically homogeneous state. The purified enzyme (estimated mol wt 83000) was optimally active at pH 6.0 and 50°C and remained active when held at 70°C (30 min) and at pH 6 to 8 for 24 h. Na⁺, Fe²⁺ and Ba²⁺ (at 5mm) enhanced enzyme activity while Hg²⁺, Zn²⁺ and Cu²⁺ (at 5mm) were inhibitory. The enzyme hydrolysed amylopectin (Km, 0.25 mg/ml), forming maltose, maltotriose and maltotetraose and hydrolyzed glycogen (Km, 0.29 mg/ml) and soluble starch (Km, 0.42 mg/ml) forming maltotriose and maltotetraose. Pullulan was not hydrolyzed.     

Two-step enzymatic synthesis of maltooligosaccharide esters

Glucose and maltose esters were synthesised in organic media by employing a lipase (E.C. 3.1.1.3) from Candida antarctica. In a second reaction step, a transglycosylation catalysed by a cyclodextrin glycosyltransferase (E.C. 2.4.1.19) from either Paenibacillus sp. F8 or Bacillus sp. strain no. 169 (DSM 2518) extended the degree of polymerisation (DP) of the carbohydrate moieties of the carbohydrate esters. The donor substrates used were either a cyclodextrin, a maltooligosaccharide or starch. The highest rate of low DP maltooligosaccharide ester formation was obtained when starch was used as glycosyl donor and caproyl maltose as glycosyl acceptor. The structures of two of the products were identified by 1H and 13C NMR and MALDI-TOF MS as capronate monoesters of maltotriose and maltotetraose, with the ester bond at C-6 of the second glucose unit from the reducing end.     

Haloalkaliphilic maltotriose-forming alpha-amylase from the archaebacterium Natronococcus sp. strain Ah-36.

A haloalkaliphilic archaebacterium, Natronococcus sp. strain Ah-36, produced extracellularly a maltotriose-forming amylase. The amylase was purified to homogeneity by ethanol precipitation, hydroxylapatite chromatography, hydrophobic chromatography, and gel filtration. The molecular weight of the enzyme was estimated to be 74,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amylase exhibited maximal activity at pH 8.7 and 55 degrees C in the presence of 2.5 M NaCl. The activity was irreversibly lost at low ionic strength. KCl, RbCl, and CsCl could partially substitute for NaCl at higher concentrations. The amylase was stable in the range of pH 6.0 to 8.6 and up to 50 degrees C in the presence of 2.5 M NaCl. Stabilization of the enzyme by soluble starch was observed in all cases. The enzyme activity was inhibited by the addition of 1 mM ZnCl2 or 1 mM N-bromosuccinimide. The amylase hydrolyzed soluble starch, amylose, amylopectin, and, more slowly, glycogen to produce maltotriose with small amounts of maltose and glucose of an alpha-configuration. Malto-oligosaccharides ranging from maltotetraose to maltoheptaose were also hydrolyzed; however, maltotriose and maltose were not hydrolyzed even with a prolonged reaction time. Transferase activity was detected by using maltotetraose or maltopentaose as a substrate. The amylase hydrolyzed gamma-cyclodextrin. alpha-Cyclodextrin and beta-cyclodextrin, however, were not hydrolyzed, although these compounds acted as competitive inhibitors to the amylase activity. Amino acid analysis showed that the amylase was characteristically enriched in glutamic acid or glutamine and in glycine.     

Purification, biochemical characterization, and gene cloning of a new extracellular thermotolerant and glucose tolerant maltooligosaccharide-forming alpha-amylase from an endophytic ascomycete Fusicoccum sp. BCC4124

An endophytic fungus, Fusicoccum sp. BCC4124, showed strong amylolytic activity when cultivated on multi-enzyme induction enriched medium and agro-industry substrates. alpha-Amylase and alpha-glucosidase activities were highly induced in the presence of maltose and starch. The purified target alpha-amylase, Amy-FC1, showed strong hydrolytic activity on soluble starch (kcat/Km=6.47 x 10(3) min(-1)(ml/mg)) and selective activity on gamma- and beta-cyclodextrins, but not on alpha-cyclodextrin. The enzyme worked optimally at 70 degrees C in a neutral pH range with t(1/2) of 240 min in the presence of Ca(2+) and starch. Maltose, matotriose, and maltotetraose were the major products from starch hydrolysis but prolonged reaction led to the production of glucose, maltose, and maltotriose from starch, cyclodextrins, and maltooligosaccharides (G3-G7). The amylase showed remarkable glucose tolerance up to 1 M, but was more sensitive to inhibition by maltose. The deduced protein primary structure from the putative gene revealed that the enzyme shared moderate homology between alpha-amylases from Aspergilli and Lipomyces sp. This thermotolerant, glucose tolerant maltooligosaccharide-forming alpha-amylase is potent for biotechnological application.     

Influence of photocrosslinking on the retrogradation of wheat starch based films

The ageing of wheat starch based materials was followed as a function of time for different systems suitably hydrated to be in the rubbery state. In particular, one aspect of retrogradation, that is to say the development of crystallinity is analysed in order to study the influence of a specific crosslinking treatment of starch films made by UV irradiation (photocrosslinking). Mechanical and thermal properties showed that an important limitation on this particular ageing occurs after photocrosslinking at room temperature. A process of accelerated ageing, monitored by modulated DSC, was also performed at 60 °C in order to increase retrogradation kinetics. The results showed a recrystallisation decrease and it appeared that the presence of the plasticiser enhances retrogradation. For a plasticised sample (17% glycerol), a decrease of 30% of the melting enthalpy was observed compared to 91% for an unplasticised one. A modification in the nature of the melting peaks, as recorded by modulated DSC, allowed us to propose a model for amylopectin retrogradation after crosslinking.     

Purification and characterization of the extracellular alpha-amylase from Streptococcus bovis JB1.

The extracellular alpha-amylase (1,4-alpha-D-glucanglucanohydrolase; EC 3.2.1.1) from maltose-grown Streptococcus bovis JB1 was purified to apparent homogeneity by ion-exchange chromatography (Mono Q). The enzyme had an isoelectric point of 4.50 and an apparent molecular mass of 77,000 Da, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was rich in acidic and hydrophobic amino acids. The 15-amino-acid NH2-terminal sequence was 40% homologous with the Bacillus subtilis saccharifying alpha-amylase and 27% homologous with the Clostridium acetobutylicum alpha-amylase. alpha-Amylase activity on soluble starch was optimal at pH 5.0 to 6.0. The enzyme was relatively stable between pH 5.5 and 8.5 and at temperatures below 50 degrees C. When soluble potato starch was used as the substrate, the enzyme had a Km of 0.88 mg.ml-1 and a kcat of 2,510 mumol of reducing sugar.min-1.mg of protein-1. The enzyme exhibited neither pullulanase nor dextranase activity and was 40 to 70% as active on amylopectin as on amylose. The major end products of amylose hydrolysis were maltose, maltotriose, and maltotetraose.     

Origins of B-type crystallinity in glycerol-plasticised, compression-moulded potato starches

The amount of B-type crystallinity in compression-moulded, glycerol-plasticised potato starches was strongly dependent on both the properties of the potato starch used and the applied processing conditions. The presence of amylose and the morphology of the potato starch used, but also processing parameters such as moulding temperature and water content during moulding affected the amount of B-type crystallinity in the materials and thus the ultimate mechanical properties of the plasticised starches. This indicated that the direct relation between composition and physical properties of processed starches is not always valid; processing parameters are important tools for controlling the physical properties of processed starches as they influence the amount of B-type crystallinity in the material. It was shown that the total amount of B-type crystallinity in the glycerol-plasticised potato starches should be considered as a summation of residual amylopectin crystallinity and recrystallisation of both amylose and amylopectin, being strongly dependent on the applied processing conditions. In order to explain the observed amount of B-type crystallinity in these starches, partial (co-)crystallisation of both amylose and amylopectin should occur at high moulding temperatures. The measured mechanical properties of the plasticised potato starches correlated well with the amount of B-type crystallinity observed in the materials.     

Purifications and Enzymatic Properties of β-Amylase and Pullulanase from Bacillus cereus var. mycoides

A β-amylase and a pullulanase produced by Bacillus cereus var. mycoides were purified by means of ammonium sulfate fractionation, adsorption on starch and celite and Sephadex G–100 column chromatography. The purified enzymes were homogeneous in disc electrophoresis.

The β-amylase released only maltose from amylose, amylopectin, starch and glycogen, and the released maltose was in β-form. The pullulanase released maltose, maltotriose and maltotetraose from β-limit dextrin and maltotriose from pullulan, but not amylose-like substance from amylopectin.

The optimum pHs of β-amylase and pullulanase were about 7 and 6~6.5, respectively. The optimum temperatures of the enzymes were about 50°C. The enzymes were inhibited by the sulfhydryl reagents such as mercuric chloride and p-chloromercuribenzoate, and the inhibitions with p-chloromercuribenzoate were restored by the addition of cysteine. The molecular weights of β-amylase and pullulanase were estimated to be 35,000±5,000 and 110,000±20,000, respectively.