An amylase gene from Drosophila pseudoobscura is expressed in Escherichia coli: Functional selection and biochemical comparisons of the fly- and clone-produced amylases

An amylase gene from Drosophila pseudoobscura was isolated from a genomic library constructed in pBR322 and cloned in Escherichia coli by selecting for the ability of its product to hydrolyze starch, a carbon source not normally utilized by E. coli. Hybridization of pAMY17F to D. pseudoobscura polytene chromosomes shows a positive signal at the amylase pseudogene locus (band 78, chromosome 3). The chimeric plasmid pAMY17F, has been altered in such a way as to increase amylase expression. Southern and Northern hybridizations to the cloned amylase DNA indicate that the source of the gene is from D. pseudoobscura. Biochemical properties such as pH optima, substrate specificities, electrophoretic analyses, inhibitor sensitivities, heat stabilities, temperature responsiveness and molecular weights indicate that the amylases produced by the fly and bacterial clone are similar and have similar properties. It appears that E. coli/pAMY17F is producing an amylase like that found in D. pseudoobscura.     

Multisubstrate specifics amylase from mushroomTermitomyces clypeatus

An amylase was purified from the culture filtrate ofTermitomyces clypeatus by ammonium sulphate precipitation, DEAE-Sephadex chromatography and gel filtration on Bio-Gel P-200 column. The electrophoretically homogeneous preparation also exhibited hydrolytic activity (in a decreasing order) on amylose, xylan, amylopectin, glycogen, arabinogalactan and arabinoxylan. The enzyme had characteristically endo-hydrolytic activity on all the substrates tested and no xylose, glucose, arabinose or glucuronic acid could be detected even after prolonged enzymatic digestion of the polysaccharides. Interestingly the enzyme had similar pH optima (5.5), temperature optima (55°C), pH stability (pH 3–10) and thermal denaturation kinetics when acted on both starch and xylan (larch wood) .K _m values were found to be 2.63 mg/ml for amylase and 6.25 mg/ml for xylanase activity. Hill’s plot also indicated that the enzyme contained a single active site for both activities. Hg²⁺ was found to be most potent inhibitor. Ca²⁺, a common activator for amylase activity, appeared to be an inhibitor for this enzyme. Thus it appeared that the enzyme had multisubstrate specificity acting as α-amylase on starch and also acting as xylanase on side chain oligosaccharides of xylan containing α-linked sugars.     

Comparative analysis of tertiary alcohol esterase activity in bacterial strains isolated from enrichment cultures and from screening strain libraries

The preparation of enantiopure tertiary alcohols is of great contemporary interest due to the application of these versatile building blocks in organic synthesis and as precursors towards high value pharmaceutical compounds. Herein, we describe two approaches taken towards the discovery of novel biocatalysts for the synthesis of these valuable compounds. The first approach was initiated with screening of 47 bacterial strains for hydrolytic activity towards the simple tertiary alcohol ester tert-butyl acetate. In conjunction, a second method focussed on the isolation of strains competent for growth on tert-butyl acetate as the sole source of carbon and energy. From functional screening, 10 Gram-positive Actinomycetes showed hydrolytic activity, whilst enrichment selection resulted in the identification of 14 active strains, of which five belong to the Gram-negative cell-wall type. Bacterial strains obtained from both approaches were viable for enantioselective hydrolysis of pyridine substituted tertiary alcohol esters in addition to bulky aliphatic and keto-derived substrates from the same class. Activity towards each of the test substrates was uncovered, with promising enantioselectivities of up to E = 71 in the hydrolysis of a para-substituted pyridine tertiary alcohol ester using a strain of Rhodococcus ruber. Interestingly strains of Microbacterium and Alcaligenes sp. gave opposite enantiopreference in the hydrolysis of a meta-substituted pyridine tertiary alcohol ester with E values of 17 and 54. These approaches show that via both possibilities, screening established strain collections and performing enrichment selection, it is possible to identify novel species which show activity towards sterically challenging substrates.     

Characterization of a recombinant amylolytic enzyme of hyperthermophilic archaeon Thermofilum pendens with extremely thermostable maltogenic amylase activity

A gene (Tpen_1458) encoding a putative alpha amylase from hyperthermophilic archaeon Thermofilum pendens (TfMA) was cloned and expressed in Escherichia coli. The recombinant amylolytic enzyme was purified by Ni-NTA affinity chromatography and its catalytic properties were examined. Purified TfMA was extremely thermostable with a half-life of 60 min at an optimal temperature of 95°C. TfMA activity increased to 136% in the presence of 5 mM CaCl₂. Maximal activity was measured toward γ-cyclodextrin with a specific activity of 56 U/mg using copper bicinchoninate method. TfMA catalyzed the ring-opening reaction by cleaving one α-1,4-glycosidic linkage of cyclodextrin to produce corresponding single maltooligosaccharide at the initial time. The final products from cyclodextrins, linear maltooligosaccharides, and starch were glucose and maltose, and TfMA could also degrade pullulan and amylase inhibitor acarbose to panose and acarviosine-glucose, respectively. These results revealed that TfMA is a novel maltogenic amylase.     

Isolation and partial characterization of three isoamylases of Rhyzopertha dominica F. (Coleoptera: Bostrichidae)

Three isoamylases of Rhyzopertha dominica (termed RdA70, RdA79, and RdA90 according to their relative mobility in gel electrophoresis) were isolated by ammonium sulfate fractionation and hydrophobic interaction chromatography. RdA70 and RdA79 showed an optimal pH of 7.0, whereas for RdA90 the optimal pH was 6.5. The three isoamylases remained stable at 50 °C for 1 h, but at 60 °C, all lost 50% of their activity in 20 min and were completely inactivated in 1 h. RdA70 and RdA79 were inhibited by albumin extracts from wheat samples varying widely in amylase inhibitory activity; however, RdA90 was highly resistant to inhibition. β-Mercaptoethanol up to 30 mM increased the activity of the three isoamylases by 2.5-fold. The action pattern of the three isoamylases was typical of endoamylases; however, differences were observed on the hydrolytic efficiency rates measured as V_max/K_m ratio on starch, amylopectin, and amylose. The hydrolyzing action of RdA90 on starch and amylopectin (V_max/K_m = 90.4 ± 2.3 and 78.9 ± 6.6, respectively) was less efficient than that on amylose (V_max/K_m = 214 ± 23.2). RdA79 efficiently hydrolyzed both amylopectin and amylose (V_max/K_m = 260.6 ± 12.9 and 326.5 ± 9.4, respectively). RdA70 hydrolyzed starch and amylose at similar rates (V_max/K_m = 202.9 ± 5.5 and 215.9 ± 6.2, respectively), but amylopectin was a poor substrate (V_max/K_m = 124.2 ± 7.4). The overall results suggest that RdA70 and RdA79 appear to belong to a group of saccharifying isoamylases that breaks down long fragments of oligosaccharide chains produced by the hydrolytic action of RdA90. The simultaneous action of the three isoamylases on starch, aside from the high resistance of RdA90 to wheat amylase inhibitors, might allow R. dominica to feed and reproduce successfully on the wheat kernel.     

An amylase gene from Drosophila pseudoobscura is expressed in Escherichia coli. Functional selection and biochemical comparisons of the fly- and clone-produced amylases

An amylase gene from Drosophila pseudoobscura was isolated from a genomic library constructed in pBR322 and cloned in Escherichia coli by selecting for the ability of its product to hydrolyze starch, a carbon source not normally utilized by E. coli. Hybridization of pAMY17F to D. pseudoobscura polytene chromosomes shows a positive signal at the amylase pseudogene locus (bank 78, chromosome 3). The chimeric plasmid pAMY17F, has been altered in such a way as to increase amylase expression. Southern and Northern hybridizations to the cloned amylase DNA indicate that the source of the gene is from D. pseudoobscura. Biochemical properties such as pH optima, substrate specificities, electrophoretic analyses, inhibitor sensitivities, heat stabilities, temperature responsiveness and molecular weights indicate that the amylases produced by the fly and bacterial clone are similar and have similar properties. It appears that E. coli/pAMY17F is producing an amylase like that found in D. pseudoobscura.     

Identification and characterization of a novel cold-adapted esterase from a metagenomic library of mountain soil

A novel lipolytic enzyme was isolated from a metagenomic library after demonstration of lipolytic activity on an LB agar plate containing 1% (w/v) tributyrin. A novel esterase gene (estIM1), encoding a lipolytic enzyme (EstIM1), was cloned using a shotgun method from a pFosEstIM1 clone of the metagenomic library, and the enzyme was characterized. The estIM1 gene had an open reading frame (ORF) of 936 base pairs and encoded a protein of 311 amino acids with a molecular mass 34 kDa and a pI value of 4.32. The deduced amino acid sequence was 62% identical to that of an esterase from an uncultured bacterium (ABQ11271). The amino acid sequence indicated that EstIM1 was a member of the family IV of lipolytic enzymes, all of which contain a GDSAG motif shared with similar enzymes of lactic acid microorganisms. EstIM1 was active over a temperature range of 1–50°C, at alkaline pH. The activation energy for hydrolysis of p-nitrophenyl propionate was 1.04 kcal/mol, within a temperature range of 1–40°C. The activity of EstIM1 was about 60% of maximal even at 1°C, suggesting that EstIM1 is efficiently cold-adapted. Further characterization of this cold-adapted enzyme indicated that the esterase may be very valuable in industrial applications.     

Isolation of a low-temperature adapted lipolytic enzyme from uncultivated micro-organism

Aims: The aim of the study was to isolate a novel lipolytic enzyme from the activated sludge of uncultured micro‐organisms. Methods and Results: The metagenomic DNA was directly extracted from the activated sludge, and a metagenomic library was constructed by using the pUC vector. The library was screened for lipolytic enzyme activity on 1% tributyrin agar plate. A clone among c. 100 000 recombinant libraries showed the lipolytic activity. The putative lipolytic gene encoding lipo1 from the metagenomic library was subcloned and expressed in Escherichia coli BL21 using the pET expression system. The expressed recombinant enzyme was purified by Ni‐nitrilotriacetic acid affinity chromatography and characterized using general substrates of lipolytic property. The gene consisted of 972 bp encoding a polypeptide of 324 amino acids with a molecular mass of 35·6 kDa. Typical residues essential for lipolytic activity such as penta‐peptide (GXSXG) and catalytic triad sequences (Ser166, Asp221 and His258) were detected. The deduced amino acid sequence of lipo1 showed low identity with amino acid sequences of esterase/lipase (32%, ZP_01528487 ) from Pseudomonas mendocina ymp and esterase (31%, AAY45707 ) from uncultured bacterium. This lipolytic enzyme exhibited the highest activity at pH 7·5 and 10°C. At thermal stability analysis, lipo1 was more unstable at 40°C than 10°C. Conclusions: An activity based strategy has been an effective method for fishing out a low‐temperature adapted lipolytic enzyme from the metagenomic library. This lipo1 enzyme can be considered to belong to the hormone‐sensitive lipase family due to the enzyme’s oxyanion hole by the sequence HGGG. Significance and Impact of the Study: Lipo1 is a novel psychrophilic esterase obtained directly from the metagenomic library. Owing its support of significant activity at low temperature, this enzyme is expected to be useful for potential application as a biocatalyst in organic chemistry.     

Optimization and immobilization of amylase obtained from halotolerant bacteria isolated from solar salterns

The objective of the present study was to isolate halotolerant bacteria from the sediment sample collected from Marakanam Solar Salterns, Tamil Nadu, India using NaCl supplemented media and screened for amylase production. Among the 22 isolates recovered, two strains that had immense potential were selected for amylase production and designated as P1 and P2. The phylogenetic analysis revealed that P1 and P2 have highest homology with Pontibacillus chungwhensis (99%) and Bacillus barbaricus (100%). Their amylase activity was optimized to obtain high yield under various temperature, pH and NaCl concentration. P1 and P2 strain showed respective, amylase activity maximum at 35 °C and 40 °C; pH 7.0 and 8.0; 1.5 M and 1.0 M NaCl concentration. Further under optimized conditions, the amylase activity of P1 strain (49.6 U mL^?1) was higher than P2 strain. Therefore, the amylase enzyme isolated from P. chungwhensis P1 was immobilized in sodium alginate beads. Compared to the free enzyme form (49.6 U mL^?1), the immobilized enzyme showed higher amylase activity as 90.3 U mL^?1. The enzyme was further purified partially and the molecular mass was determined as 40 kDa by SDS–PAGE. Thus, high activity of amylase even under increased NaCl concentration would render immense benefits in food processing industries.     

Production,purification, and characterization of two extremely halotolerant,thermostable, and alkali-stable α-amylases from Chromohalobacter sp. TVSP 101

The halophilic bacterial strain Chromohalobacter sp. TVSP 101 was shown to produce extracellular, halotolerant, alkali-stable and moderately thermophilic α-amylase activity. The culture conditions for higher amylase production were optimized with respect to NaCl, pH, temperature and substrates. Maximum amylase production was achieved in a medium containing 20% NaCl or 15% KCl at pH 9.0 and 37 °C in the presence of 0.5% rice flour and tryptone. Addition of 50 mM CaCl₂ to the medium increased amylase production by 29%. Two kinds of amylase activity, designated amylase I and amylase II, were purified from culture filtrates to homogeneity with molecular masses of 72 and 62 kDa, respectively. Both enzymes had maximal activity at pH 9.0 and 65 °C in the presence of 0–20% (w/v) NaCl but amylase I was much more stable in the absence of NaCl than amylase II. The enzymes efficiently hydrolyzed carbohydrates to yield maltotetraose, maltotriose, maltose, and glucose as the end products.     

Mixture design of starchy substrates hydrolysis by an immobilized glucoamylase from Aspergillus brasiliensis

Starch has great importance in human diet, since it is a heteropolymer of plants, mainly found in roots, as potato, cassava and arrowroots. This carbohydrate is composed by a highly-branched chain: amylopectin; and a linear chain: amylose. The proportion between the chains varies according to the botanical source. Starch hydrolysis is catalyzed by enzymes of the amilolytic system, named amylases. Among the various enzymes of this system, the glucoamylases (EC 3.2.1.3 glucan 1,4-alpha-glucosidases) are the majority because they hydrolyze the glycosidic linkages at the end of starch chains releasing glucose monomers. In this work, a glucoamylase secreted in the culture medium, by the ascomycete Aspergillus brasiliensis, was immobilized in Dietilaminoetil Sepharose-Polyethylene Glycol (DEAE-PEG), since immobilized biocatalysts are more stable in long periods of hydrolysis, and can be recovered from the final product and reused for several cycles. Glucoamylase immobilization has shown great thermal stability improvement over the soluble enzyme, reaching 66% more activity after 6?h at 60?°C, and 68% of the activity after 10 hydrolysis cycles. A simplex centroid experimental mixture design was applied as a tool to characterize the affinity of the immobilized enzyme for different starchy substrates. In assays containing several proportions of amylose, amylopectin and starch, the glucoamylase from A. brasiliensis mainly hydrolyzed the amylopectin chains, showing to have preference by branched substrates.     

Identification and characterization of novel cellulolytic and hemicellulolytic genes and enzymes derived from German grassland soil metagenomes

Soil metagenomes represent an unlimited resource for the discovery of novel biocatalysts from soil microorganisms. Three large-inserts metagenomic DNA libraries were constructed from different grassland soil samples and screened for genes conferring cellulase or xylanase activity. Function-driven screening identified a novel cellulase-encoding gene (cel01) and two xylanase-encoding genes (xyn01 and xyn02). From sequence and protein domain analyses, Cel01 (831 amino acids) belongs to glycoside hydrolase family 9 whereas Xyn01 (170 amino acids) and Xyn02 (255 amino acids) are members of glycoside hydrolase family 11. Cel01 harbors a family 9 carbohydrate-binding module, previously found only in xylanases. Both Xyn01 and Xyn02 were most active at 60°C with high activities from 4 to 10 and optimal at pH 7 (Xyn01) and pH 6 (Xyn02). The cellulase gene, cel01, was expressed in E. coli BL21 and the recombinant enzyme (91.9 kDa) was purified. Cel01 exhibited high activity with soluble cellulose substrates containing β-1,4-linkages. Activity with microcrystalline cellulose was not detected. These data, together with the analysis of the degradation profiles of carboxymethyl cellulose and barley glucan indicated that Cel01 is an endo 1,4-β-glucanase. Cel01 showed optimal activity at 50°C and pH 7 being highly active from pH range 5 to 9 and possesses remarkable halotolerance.     

Immobilization of α-amylase from mung beans (Vigna radiata) on Amberlite MB 150 and chitosan beads: A comparative study

α-Amylase from mung beans (Vigna radiata) was immobilized on two different matrices, Amberlite MB 150 and chitosan beads. Maximum immobilization obtained was 72% and 69% in case of Amberlite and chitosan beads, respectively. The pH optima of soluble α-amylase were 5.6, whereas that for immobilized amylase on chitosan and Amberlite was 7.0. Soluble amylase and Amberlite immobilized amylase showed maximum activity at 65 °C, whereas chitosan immobilized amylase showed maximum activity at 75 °C. α-Amylase immobilized on Amberlite showed apparent K_m of 2.77 mg/ml, whereas α-amylase immobilized on chitosan showed an apparent K_m of 5 mg/ml. The Amberlite-amylase and chitosan-amylase showed a residual activity of 43% and 27%, respectively, after 10 uses. The loss of activity for free amylase after 100 days of storage at 4 °C was 70%, whereas that for Amberlite- and chitosan-amylases, under the same experimental conditions, the losses were 45% and 55%, respectively. The easy availability of mung bean α-amylase, the ease of its immobilization on low-cost matrices and good stability upon immobilization in the present study makes it a suitable product for further use in industrial applications.     

Lipase-catalyzed synthesis and antibacterial activity of N-vanillylnonanamide

N-vanillylnonanamide (VAN) was successfully synthesized from vanillylamine hydrochloride by enzymatic catalysis in supercritical carbon dioxide (SC–CO₂). Five commercial lipases, Novozyme 435, Lipozyme IM, Amano PS, Amano G and Sigma Candida cylindracea type VII, as biocatalysts for VAN synthesis were compared. Lipozyme IM exhibited best yields of tested lipases. Various parameters such as time, temperature, pressure and vanillylamine hydrochloride/nonanoic anhydride ratio that influenced the reaction were investigated. Nonanoic anhydride showed the best acyl donor of the employed substrates. An amidation yield of 40% was obtained when nonanoic anhydride and Lipozyme IM were used at 170 bar and 50 °C for 23 h in SC–CO₂. Besides, addition of 2 mM divalent salts (CuCl₂ and ZnCl₂) significantly increased 11–23% yield of the VAN. The enzyme operational stability suggested that Lipozyme IM maintained over 50 °C of the initial activity for the synthesis of VAN after reuse for 69 h. Furthermore, in vitro, VAN behaved as a potential antibacterial against Escherichia coli.     

Heterologous expression of <Emphasis Type="Italic">Thermobifida fusca</Emphasis> thermostable alpha-amylase in <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> and its application in boiling stable resistant sago starch preparation

A gene encoding the thermostable α-amylase in Thermobifida fusca NTU22 was amplified by PCR, sequenced, and cloned into Yarrowia lipolytica P01g host strain using the vector pYLSC1 allowing constitutive expression and secretion of the protein. Recombinant expression resulted in high levels of extracellular amylase production, as high as 730 U/l in the Hinton flask culture broth. It is higher than that observed in P. pastoris expression system and E. coli expression system. The purified amylase showed a single band at about 65 kDa by SDS-polyacrylamide gel electrophoresis and this agrees with the predicted size based on the nucleotide sequence. About 70% of the original activity remained after heat treatment at 60°C for 3 h. The optimal pH and temperature of the purified amylase were 7.0 and 60°C, respectively. The purified amylase exhibited a high level of activity with raw sago starch. After 72-h treatment, the DP _w of raw sago starch obviously decreased from 830,945 to 237,092. The boiling stable resistant starch content of the sago starch increased from 8.3 to 18.1%. The starch recovery rate was 71%.     

Isolation and Characterization of a GDSL Esterase from the Metagenome of a Marine Sponge-associated Bacteria

Using a metagenome library constructed from a bacterial associated with a marine sponge Hyrtios erecta, we identified a novel esterase that belongs to the SGNH hydrolase superfamily of esterases. The substrate specificity of EstHE1 was determined using p-nitrophenyl (pNP) ester (C2: acetate, C4: butylate, C6: caproate, C12: laurate, C16: palmitate). EstHE1 exhibited activity against C2 (5.6 U/mg), C4 (5.1 U/mg), and C6 (2.8 U/mg) substrates. The optimal temperature for EstHE1 esterase activity of the pNP acetate substrate was 40°C, and EstHE1 retained 60% of its enzymatic activity in the 30–50°C range. This esterase showed moderate thermostability, retaining 58% of its activity even after preincubation for 12 h at 40°C. EstHE1 also maintained activity in high concentrations of NaCl, indicating that this esterase is salt-tolerant. Thus, EstHE1 has the thermal stability and salt tolerance necessary for use as an industrial enzyme.     

Molecular cloning and characterization of a new cold-active esterase from a deep-sea metagenomic library

A clone which conferred lipolytic activity at low temperature was identified from a fosmid library constructed from a South China Sea marine sediment sample. The gene responsible, estF, consisted of 1,080 bp that encoded 359 amino acid residues, with a typical N-terminal signal peptide of 28 amino acid residues. A phylogenetic analysis of amino acid sequence with other lipolytic enzymes revealed that EstF and seven closely related putative lipolytic enzymes comprised a unique clade in the phylogenetic tree. Moreover, these hypothetic esterases showed unique conservative sites in the amino acid sequence. The recombinant EstF was overexpressed and purified, and its biochemical properties were partially characterized. The optimal substrate for EstF to hydrolyze among a panel of p-nitrophenyl esters (C2 to C16) was p-nitrophenyl butyrate (C4), with a K _m of 0.46 mM. Activity quickly decreased with substrates containing an acyl chain length longer than 10 carbons. We found that EstF was active in the temperature range of 0–60°C, showed the best activity at 50°C, but was unstable at 60°C. It exhibited a high level of activity in the pH range of 7.0–10.0 showing the highest activity at pH 9.0.     

Variation in Ecological Status of Norwegian Sea Water Determined from Hydrolytic Enzyme Activities

The capacity for biopolymer transformation involving efficient and highly specific natural enzyme mechanisms was studied in seawater of the dynamic zone of the Norwegian Sea (the Voring Plateau region). Vertical and spatial variation in proteinase and amylase activities was demonstrated in seawater and the potential rates of degradation of specific substrates, azocasein and Procion-5CX-modified starch, were calculated. High proteolytic activity was demonstrated for the upper photic layer (0–10 m) in the southwestern part of the polygon (up to 88 U/l; v _pr = 7.04 mg/l/h). Proteolytic activity in the bathyal layer (1500 m and below) sharply decreased to 8–16 U/l; v _pr = 0.64–1.28 mg/l/h. Similar to other regions of the ocean, the pattern of amylase activity in seawater included low rates of polysaccharide destruction (0–4 U/l; v _st = 0–0.2 mg/l/h) in water with high proteolytic capacity and, conversely, the top amylase activity (up to 246–490 U/l; v _st 12.3–24.5 mg/l/h) in seawater layers with undetectable or low proteolytic activity. The spatial distribution of the enzyme activities can indicate the presence of waters of different origin. In the southwestern part of the polygon, statistical analysis demonstrated high correlations between hydrophysical indices (temperature, salinity, and salinity gradient) and proteinase and amylase activities. The ecological evaluation based on express enzyme-substrate tests demonstrated a stressful situation for destruction of proteins in both the photic layer and the layers below 1000 m (t _pr ≥ 10 h).__________Translated from Izvestiya Akademii Nauk, Seriya Biologicheskaya, No. 4, 2005, pp. 467–478.Original Russian Text Copyright © 2005 by Korneeva, Gordeeva, Shevchenko.     

Expression of Thermobifida fusca thermostable raw starch digesting alpha-amylase in Pichia pastoris and its application in raw sago starch hydrolysis

A gene encoding the thermostable raw starch digesting α-amylase in Thermobifida fusca NTU22 was amplified by PCR, sequenced and cloned into Pichia pastoris X-33 host strain using the vector pGAPZαA, allowing constitutive expression and secretion of the protein. Recombinant expression resulted in high levels of extracellular amylase production, as high as 510 U/l in the Hinton flask culture broth. The purified amylase showed a single band at about 65 kDa by SDS-polyacrylamide gel electrophoresis after being treated with endo-β-N-acetylglycosaminidase H, and this agrees with the predicted size based on the nucleotide sequence. About 75% of the original activity remained after heat treatment at 60°C for 3 h. The optimal pH and temperature of the purified amylase were 7.0 and 60°C, respectively. The purified amylase exhibited a high level of activity with raw sago starch. After 48-h treatment, the DPw of raw sago starch obviously decreased from 830,945 to 378,732. The surface of starch granules was rough, and some granules displayed deep cavities.