Degradation of Granular Starch by the Bacterium Microbacterium aurum Strain B8.A Involves a Modular α-Amylase Enzyme System with FNIII and CBM25 Domains

The bacterium Microbacterium aurum strain B8.A, originally isolated from a potato plant wastewater facility, is able to degrade different types of starch granules. Here we report the characterization of an unusually large, multidomain M. aurum B8.A α-amylase enzyme (MaAmyA). MaAmyA is a 1,417-amino-acid (aa) protein with a predicted molecular mass of 148 kDa. Sequence analysis of MaAmyA showed that its catalytic core is a family GH13_32 α-amylase with the typical ABC domain structure, followed by a fibronectin (FNIII) domain, two carbohydrate binding modules (CBM25), and another three FNIII domains. Recombinant expression and purification yielded an enzyme with the ability to degrade wheat and potato starch granules by introducing pores. Characterization of various truncated mutants of MaAmyA revealed a direct relationship between the presence of CBM25 domains and the ability of MaAmyA to form pores in starch granules, while the FNIII domains most likely function as stable linkers. At the C terminus, MaAmyA carries a 300-aa domain which is uniquely associated with large multidomain amylases; its function remains to be elucidated. We concluded that M. aurum B8.A employs a multidomain enzyme system to initiate degradation of starch granules via pore formation.     

Production of amylase by Arthrobacter psychrolactophilus

Arthrobacter psychrolactophilus ATCC 700733 grew with a doubling time of 1.5–2.3 h (22°C) and produced up to 0.2 units/mL (soluble starch assay) of extracellular amylase in tryptic soy broth without dextrose (TSBWD) containing 0.5% or 1.0% (w/v) soluble starch or maltose as the fermentable substrate. Time-course experiments in media containing soluble starch as substrate showed that amylolytic activity appeared in cultures at 24 h (after exponential growth had ceased), reached peak levels in 72–96 h, and declined rapidly after reaching peak levels. Peak levels were highest in TSBWD containing 1.0% soluble starch. Proteolytic activity appeared at about the same time as amylolytic activity and increased during the period of amylase production. Significant amylase production was not observed in cultures in TSBWD with 0.5% glucose or in cultures grown at 28°C, but low levels of amylase were observed in TSBWD cultures grown at 19–23°C which contained no added carbohydrate. A single band of activity was observed after electrophoresis of supernatant fractions in non-denaturing gels, followed by in situ staining for amylolytic activity. The amylase possessed a raw starch-binding domain and bound to uncooked corn, wheat or potato starch granules. It was active in the Phadebas assay for -amylase. Activity was maximum on soluble starch at a temperature between 40°C and 50°C. The amylase after purification by affinity chromatography on raw starch granules exhibited two starch-binding protein bands on SDS gels of 105 kDa and 26 kDa.     

Kinetic Analysis of Glucoamylase-Catalyzed Hydrolysis of Starch Granules from Various Botanical Sources

The kinetics of glucoamylase-catalyzed hydrolysis of starch granules from six different botanical sources (rice, wheat, maize, cassava, sweet potato, and potato) was studied by the use of an electrochemical glucose sensor. A higher rate of hydrolysis was obtained as a smaller size of starch granules was used. The adsorbed amount of glucoamylase on the granule surface per unit area did not vary very much with the type of starch granules examined, while the catalytic constants of the adsorbed enzyme (k ₀) were determined to be 23.3±4.4, 14.8±6.0, 6.2±1.8, 7.1±4.1, 4.6±3.0, and 1.6±0.6 s^?1 for rice, wheat, maize, cassava, sweet potato, and potato respectively, showing that k ₀ was largely influenced by the type of starch granules. A comparison of the k ₀-values in relation to the crystalline structure of the starch granules suggested that k ₀ increases as the crystalline structure becomes dense.     

Characterization of a Potato Starch-digesting Bacterium and Its Production of Amylase

A bacterium which can utilize potato starch granules as sole carbon source was isolated and identified as Bacillus circulans from its physiological and biochemical properties. Scanning electron microscopic observation of potato starch granules recovered from the culture broth revealed that granules were degraded gradually from their surface resulting in elongated granules with layered structures on their surface. This bacterium produced extracellular amylase which can digest potato starch granules in vitro. The amylase has a unique property in that it produces only maltohexaose from gelatinized starch in the early stage of the reaction. For the production of this amylase potato starch was found to be most effective while soluble sugars including gelatinized starch and maltose had little effect.     

Starch degradation by the mould Trichoderma viride I. The mechanism of starch degradation

The mechanism of starch degradation by the fungus Trichoderma viride was studied in strain CBS 354.44, which utilizes glucose, starch and dextrins but is unable to assimilate maltose. It was shown that the amylolytic enzyme system is completely extracellular, equally well induced by starch, amylose or amylopectin and that it consists mainly of enzymes of the glucoamylase type which yield glucose as the main product of starch hydrolysis. Small amounts of -amylase are produced also. The enzymes produced in starch cultures degrade starch, amylose and amylopectin equally well.Enzyme synthesis in starch media takes place to a considerable extent after exhaustion of the carbon source when maximum growth has been attained.Low-molecular dextrins are degraded by extracellular enzymes of the glucoamylase type. These enzymes are produced in media containing starch or dextrins. Maltotriose is consumed for only one third leaving maltose in the culture filtrate. Maltose is hardly attacked and hardly induces any amylolytic enzyme activity. No stable -glucosidase appears to be produced.     

Preparation and characterization of gelatinized granular starches from aqueous ethanol treatments

In order to modify the properties of native starch granules, the formation of gelatinized granular forms (GGS) from normal, waxy, and high amylose maize, as well as potato and tapioca starches was investigated by treating granules with aqueous ethanol at varying starch:water:ethanol ratios and then heating in a rotary evaporator to remove ethanol. The modified starches were characterized using bright field, polarized and electron microscopy. Short/long range molecular order and enthalpic transitions on heating were also studied using infrared spectroscopy, X-ray diffractometry and differential scanning calorimetry respectively. A diffuse birefringence pattern without Maltese cross was observed for most GGS samples. Treatment with aqueous ethanol resulted in starch-specific changes in the surface of granules, most noticeably swelling and disintegration in waxy maize, surface wrinkling in normal maize and tapioca, swelling and opening-up in potato starches, and swelling and bursting in high amylose maize. The ratio of ethanol to water at which original granular order was disrupted also varied with starch type. GGS had less short range molecular order than native granules as inferred by comparing 1047/1022 wave number ratio from infrared spectroscopy. Similarly, A- and B-type diffraction reflections were either reduced or completely lost with evolution of V-type patterns in GGS.     

Effect of granule size on the interference colors of starch in polarized light

Abstract

A Nikitin-Berek compensator tilted at 5.5° in a polarizing microscope was used to create a background second-order blue interference color against which starch granules were examined. A grating monochromator showed the first interference minimum of the background was at 590 nm. Starch granules have a radial molecular structure. Thus, some radii were in line with the axis of the compensator while others were across the compensator axis. Where radial birefringence counteracted the background birefringence, starch granules had two quadrants with a bright yellow first-order interference color. Where radial birefringence added to the background birefringence, there were two quadrants of second-order blue (higher than the background). In yellow quadrants where birefringence was reduced, the wavelength of the first interference minimum was reduced. In blue quadrants where birefringence was increased, the wavelength of the first interference minimum was increased. The extent to which the interference minimum of the background birefringence was shifted by starch granules was strongly dependent on the size of the starch granules. For yellow quadrants, the shifts were: r = ?0.87, P < 0.001, n = 22 for corn starch; r = ? 0.94, P <0.001, n = 22 for tapioca starch; and r = ?0.94, P <0.001, n = 12 for potato starch. For blue quadrants, the shifts were: r = 0.80, P < 0.001, n = 22 for corn; r = 0.81, P < 0.001, n = 22 for tapioca; and r = 0.93, P < 0.001, n = 16 for potato. When interference colors are used to evaluate starch granules, the granules should be similar in size or a correction must be made for granule size, and the Michel-Lévy chart of interference colors may be used to collect data subjectively.     

Digestion of Barley, Maize, and Wheat by Selected Species of Ruminal Bacteria

Differences in the digestion of barley, maize, and wheat by three major ruminal starch-digesting bacterial species, Streptococcus bovis 26, Ruminobacter amylophilus 50, and Butyrivibrio fibrisolvens A38, were characterized. The rate of starch digestion in all cereal species was greater for S. bovis 26 than for R. amylophilus 50 or B. fibrisolvens A38. Starch digestion by S. bovis 26 was greater in wheat than in barley or maize, whereas starch digestion by R. amylophilus 50 was greater in barley than in maize or wheat. B. fibrisolvens A38 digested the starch in barley and maize to a similar extent but was virtually unable to digest the starch in wheat. The higher ammonia concentration in cultures of B. fibrisolvens A38 when grown on wheat than when grown on barley or maize suggests that B. fibrisolvens A38 utilized wheat protein rather than starch. Scanning electron microscopy revealed that B. fibrisolvens A38 initially colonized cell wall material, while S. bovis 26 randomly colonized the endosperm and R. amylophilus 50 preferentially colonized starch granules. There was subsequent colonization but only superficial digestion of wheat starch granules by B. fibrisolvens A38. Variation in the association between starch and protein within the endosperm of cereal grains contributes to the differential effectiveness with which amylolytic species can utilize cereal starch.     

Determination of hydrophobicity of dry-heated wheat starch granules using sucrose fatty acid esters (SFAE)

Sucrose fatty acid esters (SFAE) were adsorbed onto dry-heated (120?°C for 10, 20, 40, 60, and 120?min) wheat starch granules and extracted with ethyl ether in a Soxhlet apparatus without gelatinization of the starch granules. The amount of sucrose in the extracted SFAE was determined by the phenol sulfate method. A gradual increase of the sucrose from 159 to 712?μg, in SFAE per gram of starch, occurred with increasing dry-heating time and demonstrated the increased hydrophobicity of the starch granules. Increase of the SFAE was highly correlated (r?=?0.9816) to increase of the oil-binding capacity of the dry-heated wheat starch granules. Non-waxy rice, waxy rice, sweet potato, and potato starch granules also showed higher hydrophobicity after dry-heating by this method.     

Determination of the maximum water solubility of eight native starches and the solubility of their acidic-methanol and -ethanol modified analogues

The maximum water solubilities of eight native starches from potato, shoti, tapioca, maize, waxy maize, amylomaize-7, wheat, and rice and their acid-methanol and acid-ethanol modified analogues have been determined. Maximum solubilities of 18.7 and 17.4 mg/mL were obtained for waxy maize and tapioca and 12.4 mg/mL for potato and maize starches by autoclaving 220 mg/10 mL at 121 degrees C; 8.7 mg/mL was obtained for shoti starch by stirring in 85:15 (v/v) Me(2)SO-H(2)O at 20 degrees C; and 7.0 and 5.2mg/mL for rice and amylomaize-7 starches by stirring in 1M NaOH at 20 degrees C. The acid-alcohol treated starches were 4-9 times more soluble than their native starches. The compositions of the solubilized starches had, in general, much higher ratios of amylose to amylopectin than the ratios in their native granules. A major exception to this was the acid-methanol treated potato, shoti, and rice starches that had much lower ratios of amylose to amylopectin than the ratios in their granules.     

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     

Characterization of a Granule-Bound Starch Synthase Isoform Found in the Pericarp of Wheat

Waxy wheat (Triticum aestivum L.) lacks the waxy protein, which is also known as granule-bound starch synthase I (GBSSI). The starch granules of waxy wheat endosperm and pollen do not contain amylose and therefore stain red-brown with iodine. However, we observed that starch from pericarp tissue of waxy wheat stained blue-black and contained amylose. Significantly higher starch synthase activity was detected in pericarp starch granules than in endosperm starch granules. A granule-bound protein that differed from GBSSI in molecular mass and isoelectric point was detected in the pericarp starch granules but not in granules from endosperm. This protein was designated GBSSII. The N-terminal amino acid sequence of GBSSII, although not identical to wheat GBSSI, showed strong homology to waxy proteins or GBSSIs of cereals and potato, and contained the motif KTGGL, which is the putative substrate-binding site of GBSSI of plants and of glycogen synthase of Escherichia coli. GBSSII cross-reacted specifically with antisera raised against potato and maize GBSSI. This study indicates that GBSSI and GBSSII are expressed in a tissue-specific manner in different organs, with GBSSII having an important function in amylose synthesis in the pericarp.     

A new insight into the gelatinization process of native starches

The gelatinization characteristics of seven different food starches (regular corn, high-amylose corn, waxy corn, wheat, rice, potato, and tapioca) were investigated. Each starch sample type was heated to 35, 40, 45, etc. up to 85 °C at 5 °C intervals, and freeze-dried. The treated samples were analyzed using light microscopy, scanning electron microscopy (SEM), differential scanning calorimetry (DSC), X-ray diffraction (XRD), and high-performance size exclusion chromatography (HPSEC). When heated, granules underwent structural changes prior to the visible morphological changes that took place during gelatinization. The nature of these structural changes depended on starch type. These results indicate that the starch gelatinization process is more complex than a simple granular order-to-disorder transition.     

Deciphering kas operon locus in Mycobacterium aurum and genesis of a recombinant strain for rational-based drug screening

Aims: To generate a recombinant Mycobacterium aurum strain for screening of antimycobacterial compounds affecting fatty acid synthase type II (FAS-II) elongation pathway. Methods and Results: kas operon locus was delineated in M. aurum, a fast growing nonpathogenic strain. Cloning and sequencing all the genes of the operon showed similar organization and sequence similarities with Mycobacterium tuberculosis (H37Rv) orthologues. Further, we cloned the upstream region of M. tuberculosis kas operon in fusion with lacZ reporter gene and put it in M. aurum. Recombinant M. aurum strain showed continued expression of reporter gene throughout the growth while an increased expression of the reporter gene was noticed only after treatment with FAS-II pathway inhibitors. Swapping of the regulatory sequence aborts the increased reporter gene expression after same antibiotic treatments. Conclusions: kas operon genes are similarly organized in M. tuberculosis and M. aurum. H37Rv kas operon promoter upregulates the reporter gene expression in M. aurum only upon treatment with drugs inhibiting FAS-II pathway. Significance and Impact of the Study: It would serve as a good second-line screen for characterization of compounds showing antimycobacterial activity in a first-line screen. With the simplicity of β-galactosidase enzyme assay the system can be easily adapted in high-throughput mode.     

The influence of tapioca on the growth,the activity of glucoamylase and pigment production of <Emphasis Type="Italic">Monascus purpureus</Emphasis> UKSW 40 in soybean-soaking wastewater

The present study evaluates the usefulness of tapioca starch as additional carbon source for the growth of Monascus purpureus in soybean-soaking wastewater (SSW). The result revealed that M. purpureus grown on 2.0% (w/v) tapioca starch in SSW produced significantly (P < 0.05) higher amounts of biomass and production of the pigments (OD₄₀₀ and OD₅₀₀) when compared to those grown on glucose-or maltose-containing media. However, the glucoamylase activity of M. purpureus grown on the tapioca-SSW medium was not significantly increased when compared to those from the glucose-containing medium.     

Molecular cloning and expression of an extracellular α-amylase gene from an Antarctic deep sea psychrotolerant <Emphasis Type="Italic">Pseudomonas stutzeri</Emphasis> strain 7193

Psychrotolerant Pseudomonas stutzeri strain 7193 capable of producing an extracellular α-amylase was isolated from deep sea sediments of Prydz Bay, Antarctic. The 59678-Da protein (AmyP) was encoded by 1665-bp gene (amyP). The deduced amino acid sequence was identified with four regions, which are conserved in amylolytic enzymes and form a catalytic domain, and was predicted to be maltotetraose forming extracellular amylase by using the I-TASSER online server. Purification of AmyP amylases from both the recombinant of Escherichia coli Top 10 F′ and strain 7193 was conducted. Biochemical characterization revealed that the optimal amylase activity was observed at pH 9.0 and temperature 40°C. The enzymes were unstable at temperatures above 30°C, and only retain half of their highest activity after incubation at 60°C for 5 min. Thin-layer chromatography analysis of the products of the amylolytic reaction showed the presence of maltotetraose, maltotriose, maltose and glucose in the starch hydrolysate.     

Role of the pectinolytic enzyme in the lactic acid fermentation of potato pulp by<Emphasis Type="Italic"> Rhizopus oryzae</Emphasis>

Rhizopus oryzae strain NBRC 4707 produced lactic acid and ethanol more efficiently than strain NRRL 395 in potato pulp, an agricultural by-product of the starch industry. The two strains developed comparable activities of xylanase, cellulase, -amylase, and glucoamylase, while the polygalacturonase activity of strain NBRC 4707 was double that of strain NRRL 395. The addition of commercial pectinase enhanced the formation of metabolites, suggesting that the degradation of pectic substances determines the fermentation of potato pulp by R. oryzae. Orange and apple peel were more effective in the induction of polygalacturonase activity than potato pulp, sugarbeet pulp, or wheat bran when used as a principal carbon source for fungal growth in a solid-state culture. The fungal cells in both types of fruit peel stimulated the fermentation of potato pulp and increased the quantity of lactic acid and ethanol to higher levels than those in other agricultural by-products.     

Production of an amylase-degrading raw starch by Gibberella pulicaris

An endophytic fungus, Gibberella pulicaris, produced an amylase which degraded raw starches from cereals and other crops including raw potato, sago, tapioca, corn, wheat and rice starch. In each case, glucose was the main product. Among the raw starches used, raw potato starch gave the highest enzyme activity (85 units mg^–1 protein) and raw wheat starch the lowest (49 units mg^–1 protein). The highest amylase production (260 units mg^–1 protein) was achieved when the concentration of raw potato starch was increased to 60 g l^–1. Optimum hydrolysis was at 40°C and pH 5.5.     

Isolation and physiological study of an amylolytic strain of Lactobacillus plantarum

Summary An amylolytic lactic acid bacterium identified as Lactobacillus plantarum was isolated from cassava roots (Manihot esculenta var. Ngansa) during reting. The amylolytic enzyme synthesized was an extracellular -amylase with an optimum pH of 5.0 and an optimum temperature of 55° C. Cultured on starch, the strain displayed a growth rate of 0.43 h^–1, a biomass yield of 0.19 g·g^–1 and a lactate yield of 0.81 g·g^–1. The growth kinetics were similar on starch and glucose. Sufficient enzyme was synthesized and starch hydrolysis was not a limiting factor for growth. Biosynthesis of the enzyme was observed when the glucose concentration was less than 6.7 g·l^–1 and reached up to 4 IU·ml^–1 at the end of the fermentation. Offprint requests to: M. Raimbault     

Amylolytically-resistant tapioca starch modified by combined treatment of branching enzyme and maltogenic amylase

Tapioca starch was modified using branching enzyme (BE) isolated from Bacillus subtilis 168 and Bacillus stearothermophilus maltogenic amylase (BSMA), and their molecular fine structure and susceptibility to amylolytic enzymes were investigated. By BE treatment, the molecular weight decreased from 3.1 × 10⁸ to 1.7 × 10⁶, the number of shorter branch chains (DP 6–12) increased, the number of longer branch chains (DP >25) decreased, and amylose content decreased from 18.9% to 0.75%. This indicated that α–1,4 linkages of amylose and amylopectin were cleaved, and moiety of glycosyl residues were transferred to another amylose and amylopectin to produce branched glucan and BE-treated tapioca starch by forming α–1,6 branch linkages. The product was further modified with BSMA to produce highly-branched tapioca starch with 9.7% of extra branch points. When subject to digestion with human pancreatic α-amylase (HPA), porcine pancreatic α-amylase (PPA) and glucoamylase, highly-branched tapioca starch gave significantly lowered α-amylase susceptibility (7.5 times, 14.4 times and 3.9 times, respectively), compared to native tapioca starch.