Thermostable Α-amylase Research Articles

Cassava Pulp as a Biofuel Feedstock of an Enzymatic Hydrolysis Proces

Cassava pulp, a low cost solid byproduct of cassava starch industry, has been proposed as a high potential ethanolic fermentation substrate due to its high residual starch level, low ash content and small particle size of the lignocellulosic fibers. As the economic feasibility depends on complete degradation of the polysaccharides to fermentable glucose, the comparative hydrolytic potential of cassava pulp by six commercial enzymes were studied. Raw cassava pulp (12% w/v, particle size <320 µm) hydrolyzed by both commercial pectinolytic (1) and amylolytic (2) enzymes cocktail, yielded 70.06% DE. Hydrothermal treatment of cassava pulp enhanced its susceptibility to enzymatic cleavageas compared to non-hydrothermal treatment raw cassava pulp. Hydrothermal pretreatment has shown that a glucoamylase (3) was the most effective enzyme for hydrolysis process of cassava pulp at temperature 65 °C or 95 °C for 10 min and yielded approximately 86.22% and 90.18% DE, respectively. Enzymatic pretreatment increased cassava pulp vulnerability to cellulase attacks. The optimum conditions for enzymatic pretreatment of 30% (w/v) cassava pulp by a potent cellulolytic/ hemicellulolytic enzyme (4) was achieves at 50 °C for 3, meanwhile for liquefaction and saccharification by a thermo-stable α-amylase (5) was achieved at 95 °C for 1 and a glucoamylase (3) at 50 °C for 24 hours, respectively, yielded a reducing sugar level up to 94,1% DE. The high yield of glucose indicates the potential use of enzymatic-hydrothermally treated cassava pulp as a cheap substrate for ethanol production.

Impact of an acid fungal protease in high gravity fermentation for ethanol production using indian sorghum as a feedstock

This study evaluated the conventional jet cooking liquefaction process followed by simultaneous saccharification and fermentation (SSF) at 30% and 35% dry solids (DS) concentration of Indian sorghum feedstock for ethanol production, with addition of acid fungal protease or urea. To evaluate the efficacy of thermostable α-amylase in liquefaction at 30% and 35% DS concentration of Indian sorghum, liquefact solubility, higher dextrins, and fermentable sugars were analyzed at the end of the process. The liquefact was further subjected to SSF using yeast. In comparison with urea, addition of an acid fungal protease during SSF process was observed to accelerate yeast growth (μ), substrate consumption (Q(s)), ultimately ethanol yield based on substrate (Y(p/s)) and ethanol productivity based on fermentation time (Q(p)). The fermentation efficiency and ethanol recovery were determined for both concentrations of Indian sorghum and found to be increased with use of acid fungal protease in SSF process.

Extrusion with Thermostable α-amylase Injection as Pretreatment Method for Ethanol Production from Corn Starch

Pretreatment technologies have been developed to increase the bioconversion rate of biomass into fermentable sugar. The objective of this research was to investigate extrusion with thermostable α-amylase injection at different melt temperatures 95, 115 and 135°C on functional properties, ethanol content and conversion (%) of corn starch extrudates. Saccharomyces cerevisae (ATCC 24858) was used for ethanol production. In the present study, significant increase in ethanol production was achieved by the injection of thermostable α-amylase during extrusion process at melt temperature 115°C. The data clearly showed that thermostable α-amylase injection gave significantly increased (p<0.05) ethanol content at melt temperature 115°C from fermentation period from 24 to 48 hr. Industrial bio-ethanol production by direct fermentation following extrusion with thermostable α-amylase injection and omitting the saccharification step will be very effective in reducing ethanol production costs in countries like U.S. Therefore, ethanol production from extruded corn starch with thermostable α-amylase injection is a significant finding that could be applied to improve bioconversion rate for ethanol production.

Simultaneous Saccharification of Corn Starch in Gluconic Acid Production by Aspergillus niger Immobilized on Nonwoven Fabric in a Pressurized Reactor

Simultaneous saccharification of corn starch and fermentation to gluconic acid (SSF) was examined with Aspergillus niger immobilized on nonwoven fabric under high dissolved oxygen (DO). The starch liquefaction step to maltodextrins by the action of thermostable α-amylase was optimized using the dextrose equivalent value as an index. SSF was carried out using liquefied starch with simultaneous addition of commercial amyloglucosidase, and A. niger for gluconic acid production at DO of 150 mg/l by supplying pressurized oxygen gas. Treatment with α-amylase at 86oC, followed by amyloglucosidase at 30oC resulted in nearly 100% saccharification when using 300 g/l corn starch. Under conditions optimized for the saccharification steps, successful repeated batch production of gluconic acid of 272 g/l with space–time yield of 6.1 g/l/h was achieved with higher than 90% production yield from corn starch without significant loss of production activity for 450 h.

Purification and enzymatic identification of an acid stable and thermostable α-amylase fromRhizopus microsporus

Rhizopus microsporus, recently isolated from a solid culture of Heng-Shui Lao-Bai-Gan (HSLBG, a famous distilled liquor in Northern China) was found to produce a novel extracellular acid stable and thermostable α-amylase. This fungal α-amylase was purified using ammonium precipitation, Sephadex G-25 desalination and DEAE-52 cellulose chromatography. Its molecular weight was estimated to be 75 kDa by SDS–PAGE. The optimum pH and temperature of this enzyme was pH 5.0 and 70°C respectively. Thermostability and kinetic analysis through the Arrhenius and Michaelis–Menten equations revealed that this enzyme showed an exceptional activity at low pH and high temperature. A combination of this thermostability and acid stability could be a valuable trait for the efficient hydrolysis of amylose to glucose in large-scale biotechnology applications. Copyright © 2012 The Institute of Brewing & Distilling

Improving the thermostability of alpha-amylase by combinatorial coevolving-site saturation mutagenesis

BackgroundThe generation of focused mutant libraries at hotspot residues is an important strategy in directed protein evolution. Existing methods, such as combinatorial active site testing and residual coupling analysis, depend primarily on the evolutionary conserved information to find the hotspot residues. Hardly any attention has been paid to another important functional and structural determinants, the functionally correlated variation information--coevolution.ResultsIn this paper, we suggest a new method, named combinatorial coevolving-site saturation mutagenesis (CCSM), in which the functionally correlated variation sites of proteins are chosen as the hotspot sites to construct focused mutant libraries. The CCSM approach was used to improve the thermal stability of α-amylase from Bacillus subtilis CN7 (Amy7C). The results indicate that the CCSM can identify novel beneficial mutation sites, and enhance the thermal stability of wild-type Amy7C by 8°C ( ), which could not be achieved with the ordinarily rational introduction of single or a double point mutation.ConclusionsOur method is able to produce more thermostable mutant α-amylases with novel beneficial mutations at new sites. It is also verified that the coevolving sites can be used as the hotspots to construct focused mutant libraries in protein engineering. This study throws new light on the active researches of the molecular coevolution.

Production of a Thermostable Α-Amylase and its Assay using &lt;em&gt;Bacillus Licheniformis&lt;/em&gt; Isolated from Excavated Land Sites in Ibadan, Nigeria

Screening for amylolytic properties from obtained isolates was carried out on starch agar plates while production and characterization of &#945;-amylase was carried out using submerged fermentation. The isolated organism was identified as Bacillus licheniformis. Physiological studies on the isolate showed that temperature of 55&#186;C and pH 7 was optimum for growth of the organism while fructose (1%), peptone (0.5%), pH 7 and temperature of 60&#186;C supported optimum amylase production. Crude characterization of &#945;-amylase revealed optimum amylolytic activity at pH 7 and a temperature of 70&#186;C. The isolated Bacillus licheniformis produced thermostable &#945;-amylase with characteristics suitable for use in starch processing other industries.

Production of Acid-Stable and High-Maltose-Forming α-Amylase of Bacillus acidicola by Solid-State Fermentation and Immobilized Cells and Its Applicability in Baking

Among matrices used for immobilizing Bacillus acidicola cells [calcium alginate, chitosan + alginate, scotch brite, and polyurethane foam (PUF)], α-amylase production was highest by PUF-immobilized cells (9.1U ml(-1)), which is higher than free cells (7.2U ml(-1)). The PUF-immobilized cells could be reused over seven cycles with sustained α-amylase production. When three variables (moisture, starch, and ammonium sulfate), which significantly affected enzyme production in solid-state fermentation (SSF), were optimized using response surface methodology, 5.6-fold enhancement in enzyme production was attained. The enzyme production in SSF is 3.8-fold higher than that in submerged fermentation. The bread made by supplementing dough with α-amylase of B. acidicola scored better than those with the xylanase of Bacillus halodurans and thermostable α-amylase of Geobacillus thermoleovorans.

Stimulation of thermal stability of α-amylase from Bacillus icheniformis ATCC 6346 by treating with cations

α-Amylases (1,4-α-D-glucan glucanohydrolase; E.C.3.2.1.1) catalyze the cleavage of α-1,4-glucosidic linkages in starch, glycogen, and various oligosaccharides. Thermostable α-amylases from Bacillus species are of great industrial importance in the production of corn syrup or dextrose. In this study effect of different cations on the enhancement of stability of α-amylase from Bacillus licheniformis ATCC 6346 was examined. Optimal activity of the enzyme was at pH 7.0 and 85 °C. α-Amylase activity was strongly inhibited by Cu2+, Hg2+ and Mn2+ but less affected by Mg2+ and Ba2+. Ca2+ and Na+ stimulated the enzyme activity at 85 °C and at pH 7.0. Addition of 0.01 M Na+ enhanced the enzyme stability from 1-33% for 60 min at 85 °C and pH 7.0. With 0.1M Na+, 100 % of initial enzyme activity was retained for 150 min and 70 min at 60 °C and 70 °C, respectively and 88% activity was retained at 80 °C, at pH 7.0 for 60 min. In the presence of 1 mM Ca2+, no loss of activity was observed in 60 min, at 85 °C and pH 7.0. Combined addition of 1mM Ca2+ and 0.1 M Na+, retained 17.3 % of the enzyme activity for180 min. But the enzyme in the presence of 1 mM Ca2+ and 0.1 M Na+ separately, lost its total activity in 120 min and 90 min, respectively at 95 °C and pH 7.0. DOI: http://dx.doi.org/10.4038/cjsbs.v41i1.4536 Ceylon Journal of Science (Bio. Sci.) 41 (1): 35-44, 2012

The stability and thermodynamic parameters of a very thermostable and calcium-independent α-amylase from a newly isolated bacterium, Anoxybacillus beppuensis TSSC-1

Anoxybacillus beppuensis TSSC-1 (GenBank Number, EU710556), a thermophilic bacterium isolated from a hot spring reservoir, was found to optimally secrete a monomeric α-amylase at 55°C and pH 7. The enzyme was purified to homogeneity by a single-step purification on phenyl sepharose 6FF, achieving a 58% yield, 10,000U/mg specific activity and 19.5 fold purification. The molecular weight, Km and Vmax were 43kD, 0.5mgml−1 and 3571.42μmolml−1m−1, respectively. The enzymatic catalysis of soluble starch was optimum at 80°C and pH 7. The thermodynamic parameters, Kd, t1/2, ΔH*, ΔS*, E and ΔG*, were consistent. The very compact structure of the enzyme and the transitional enzyme–substrate complex resisted denaturation at extreme temperatures and alkaline pH. The Kd and t1/2 measurements were consistent with the high thermostability and pH tolerance observed. The structural stability of the enzyme was also reflected by the values of ΔH*, ΔS*, E and ΔG*. While the enzyme did not exhibit metal ion dependency, it was resistant to chemical denaturation. The broad thermo- and pH-tolerance of this enzyme suggests potential commercial opportunities.

Cloning and characterization of two new thermostable and alkalitolerant α-amylases from the Anoxybacillus species that produce high levels of maltose

Two genes that encode α-amylases from two Anoxybacillus species were cloned and expressed in Escherichia coli. The genes are 1,518 bp long and encode 506 amino acids. Both sequences are 98% similar but are distinct from other well-known α-amylases. Both of the recombinant enzymes, ASKA and ADTA, were purified using an α-CD-Sepharose column. They exhibited an optimum activity at 60°C and pH 8. Both amylases were stable at pH 6-10. At 60°C in the absence of Ca²⁺, negligible reduction in activity for up to 48 h was observed. The activity half-life at 65°C was 48 and 3 h for ASKA and ADTA, respectively. In the presence of Ca²⁺ ions, both amylases were highly stable for at least 48 h and had less than a 10% decrease in activity at 70°C. Both enzymes exhibited similar end-product profiles, and the predominant yield was maltose (69%) from starch hydrolysis. To the best of our knowledge, most α-amylases that produce high levels of maltose are active at an acidic to neutral pH. This is the first report of two thermostable, alkalitolerant recombinant α-amylases from Anoxybacillus that produce high levels of maltose and have an atypical protein sequence compared with known α-amylases.

세포외 고온성 α-아밀라제를 생산하는 Alicyclobacillus acidocaldarius 의 분리 및 효소생산용 최적 배양 조건

A thermophilic Alicyclobacillus acidocaldarius, which produces thermostable α-amylase, was isolated from the hot water effluent of a boiled rice mill near Tirupati, Andhra Pradesh, India. The effect of different culture conditions on the growth and production of extracellular α-amylase by thermophilic A. acidocaldarius was investigated in laboratory scale. The results showed that the optimum conditions for the production of α-amylase are a temperature of 60°C, pH of 6.0, and medium starch concentration of 1.0%, and yeast extract and tryptone of 0.2%. Surfactants, like Tween-20 and SDS, up to 0.02%, were found to increase the bacterial growth and enzymes. Further increase in their concentration resulted in significantly decreased enzyme production.

Density functional theory (DFT) outlook of biological active site junction to single walled carbon nano tube

The α-amylases are required for the break of starch into oligosaccharides. Because starch has the most solubility at high temperatures, thermo stable α-amylase play a key role in industrial hydrolysis. The temperature effects on not only the enzyme structure, but also enzyme activity. The bacillus licheniformis itself is not a thermopile but it contains a rather thermo stable α-amylase that is the most widely used as α-amylase in the starch industry. In the present study, we made structure of nano tubeactive site of this enzyme and focused mainly on the thermo stability. On the other hand, in this theoretical study, we studied the influence of the nano tube on the relative stability of enzyme and this information was compared with experimental data. At first, nano tube–active site of α-amylase was optimized then Quantum chemical calculations were performed by the GAUSSIAN 98 program. IR parameters consisting thermo chemical parameters were calculated on the optimized structure. Our results clearly indicated that in comparison with experimental investigations, nano tube play an important role in the enzyme's thermo stability, so that the most stable condition can be observed in the case of nano tube- enzyme.

Effect of partial pressure of CO2 on the production of thermostable α-amylase and neutral protease by Bacillus caldolyticus

Controlling the concentration of dissolved oxygen is a standard feature in aerobic fermentation processes but the measurement of dissolved CO2 concentrations is often neglected in spite of its influence on the cellular metabolism. In this work room air and room air supplemented with 5% and 10% carbon dioxide were used for aeration during the cultivation of the thermophilic microorganism Bacillus caldolyticus (DSM 405) on starch to produce alpha-amylase (E.C. 3.2.1.1) and neutral protease (E.C. 3.4.24.27/28). The increased CO2 concentrations resulted in a 22% raise in activity of secreted alpha-amylase and a 43% raise in protease activity when compared with aeration with un-supplemented room air. There was no effect on the final biomass concentration. Furthermore, the lag-phase of fermentation was reduced by 30%, further increasing the productivity of alpha-amylase production. Determinations of dissolved CO2 in the culture broth were conducted both in situ with a probe as well as using exhaust gas analysis and both the methods of quantification showed good qualitative congruence.

Isolation of a thermophilic Anoxybacillus flavithermus sp. nov. and production of thermostable α-amylase under solid-state fermentation (SSF)

A new bacteria was isolated from hot-spring water of Gazligol, Afyonkarahisar in Turkey. Based on morphological and biochemical tests and 16S rRNA gene sequence analysis, the isolate belonged to the Anoxybacillus flavithermus species which has 99% similarity with the bacterium DNA. The production of α-amylase by thermophilic Anoxybacillus flavithermus was investigated under solid-state fermentation by using some agricultural waste as substrates. Solid substrates such as rice husk, banana husk, millet, water melon husk, lentil bran, wheat bran and maize oil cake were studied for enzyme production. Of these, rice husk was proved as the best substrate for α-amylase production (1,271 U/mg). The maximum α-amylase production was observed as 1,803 U/mg at 72 h, 1,000 μm particle size, 70% initial moisture content (w/v), and 40% inoculum level (v/w). Among the various nitrogen sources tested, 1% peptone (3,170 U/mg) was found to be the best nitrogen source for α-amylase production. As additional carbon sources, 1% starch (2,364 U/mg) enhanced α-amylase production. The optimum temperature for the activity of α-amylase was found to be 70°C. The enzyme was optimally active at pH 6.0 and stable in the pH range of 6.0–8.0.

Fatal Imperfection of Enzymatic-HPLC Quantitative Method for Non-Digestible Oligosaccharides and Its Proposed Solution Strategy Newly Quantitative Method for Non-Digestible Oligosaccharides

The enzymatic-HPLC quantitative method is widely used for the determination of non-digestible oligosaccharide. However, the results obtained from the method are inconsistent with some reports. In the present study, we clarified the reason that the existent enzymatic-HPLC method could not accurately measure the quantity of non-digestible oligosaccharide, and then we developed the improved enzymatic-HPLC method for accurate quantification of non-digestible oligosaccharide. When some digestible and non-digestible oligosaccharides were measured by the existent enzymatic-HPLC method, 67% of isomaltooligosaccharide (IMO) was recovered as intact form, and 91% of sucrose and 95% of lactose were recovered as intact form, respectively. This result is questionable because IMO, sucrose and lactose were clearly measured as non-digestible oligosaccharides in this method. However, IMO was readily hydrolyzed and the intact form was not detected by the improved enzymatic-HPLC method which pig small intestinal enzymes were added to the hydrolyzing process in the existent enzymatic-HPLC method. Also, sucrose and lactose were completely hydrolyzed and the intact form were not recovered. The recovery rates of intact fructooligosaccharide (FOS) and resistant maltodextrin (RMD), which are not digested by small intestinal disaccharidase, were 92.1% and 78.8%, respectively. The contradiction appears to demonstrate that thermostable α-amylase and amyloglucosidase may not be able to hydrolyze the low molecular weight oligosaccharides, although they can hydrolyze spontaneously high molecular polysaccharides. In conclusion, the present study strongly suggests that the existent enzymatic-HPLC quantitative method cannot accurately measure non-digestible oligosaccharides, and that the improved enzymatic-HPLC method is suitable for the quantity of oligosaccharides. Keywords: Non-digestible oligosaccharide, enzymatic-HPLC method, isomaltooligosaccharide, pig small intestinal enzymes, quantitative method, AOAC method, dietary fiber, ructooligosaccharide, oligosaccharides, Enzyme, ethanol, amyloglucosidase, polysaccharides, disaccharides,, oligomers, galactosyl-sucrose, calcium, magnesium

Purification and characterization of a thermostable α-amylase from Geobacillus stearothermophilus

Purification and characterization of a thermostable α-amylase from Geobacillus stearothermophilus

Composition of structural carbohydrates in biomass: Precision of a liquid chromatography method using a neutral detergent extraction and a charged aerosol detector

We adapted and optimized a method to quantify the cellulose, hemicellulose, xylan, arabinan, mannan, galactan contents in lignocellulosic biomass. This method is based on a neutral detergent extraction (NDE) of the interfering biomass components, followed by a sulfuric acid hydrolysis (SAH) of the structural polysaccharides, and a liquid chromatography with charged aerosol detection (LC-CAD) to analyze the released monosaccharides. The first step of this NDE-SAH-LC-CAD method aims at removing all compounds that interfere with the subsequent sulphuric acid hydrolysis or with the subsequent chromatographic quantification of the cellulosic and hemicellulosic monosaccharides. This step includes starch hydrolysis with an analytical thermostable α-amylase followed by an extraction of soluble compounds by a Van Soest neutral detergent solution (NDE). The aim of this paper was to assess the precision of this method when choosing fiber sorghum (Sorghum bicolor (L.) Moench), tall fescue (Festuca arundinacea Schreb.) and fiber hemp (Cannabis sativa L.) as representative lignocellulosic biomass. The cellulose content of fiber sorghum, tall fescue and fiber hemp determined by the NDE-SAH-LC-CAD method were 28.7±1.0, 29.7±1.0 and 43.6±1.2g/100g dry matter, respectively, and their hemicellulose content were 18.6±0.5, 16.5±0.5 and 14.5±0.2g/100g dry matter, respectively. Cellulose, mannan and galactan contents were higher in fiber hemp (dicotyledon) as compared to tall fescue and fiber sorghum (monocotyledons). The xylan, arabinan and total hemicellulose contents were higher in tall fescue and fiber sorghum as compared to fiber hemp. The precision of the NDE-SAH-LC-CAD method was better for polysaccharide concentration levels above 1g/100g dry matter. Galactan analysis offered a lower precision, due to a lower CAD response intensity to galactose as compared to the other monosaccharides. The dispersions of the results (expanded uncertainty) of the NDE-SAH-LC-CAD method were smaller as compared to the Van Soest (VS) method. In addition, the NDE-SAH-LC-CAD method was able to provide additional information on the composition of the hemicellulose (xylan, arabinan, mannan and galactan content) that is not provided by the Van Soest method. The NDE-SAH-LC-CAD method offers also the advantage of a better specificity for hemicellulose and cellulose, as compared to the NREL and Uppsala methods.

Purification and biochemical characterization of a Ca 2+ - independent, thermostable and acidophilic α-amylase from Bacillus sp. RM16

Bacillus sp. RM16 was isolated from a hot spring in Karachi and screened for the production of α- amylase. The enzyme was obtained after 72 h cultivation of strain in Luria broth containing 1% starch (w/v). Enzyme Amy RM16 was purified to electrophoretic homogeneity by a series of sequential steps including precipitation with ammonium sulfate at 70% saturation, Q-Sepharose, Phenyl Sepharose and reversed phase chromatography. The purified enzyme is made up of a single polypeptide chain of 66 kDa as established by a combination of SDS-PAGE and zymographic analysis. In our experimental conditions, a total yield of 1.35% with specific activity of 6380U/mg was obtained providing 17 fold final purification of the enzyme. Biochemical characterization of the Amy RM16 such as optimum temperature and pH, substrate specificity and enzymatic susceptibilities towards different metal ions and inhibitors were also performed. Results of these studies revealed that, the enzyme is active at wide temperature range with optimum activity at 80°C and retained 85% of the activity for 3 h at 50°C and around 50% of remaining activity for 1 h at 80°C. The enzyme showed optimum activity at pH 5.0. On the other hand, Ca +2 and EDTA (1 to 5 mM) did not significantly affect the enzyme activity. The main substrate for the enzyme was found to be starch but it could also hydrolyze raw starch, dextrin, γ-cyclodextrin and pullulan. Key words: Ca 2+ -independent, Bacillus sp, thermostable α-amylases, low pH profile, enzyme, raw starch digestion, HPLC.

Purifikasi dan Karakterisasi α-amilase Termostabil dari Bacillus stearothermophilus TII-12

&lt;p&gt;Purification and Characterization of Thermostable&lt;br /&gt;α-amylase from Bacillus stearothermophilus TII-12. Puji&lt;br /&gt;Lestari, Nur Richana, Abdul A. Darwis, Khaswar Syamsu,&lt;br /&gt;and Untung Murdiyatmo. Thermostable α-amylase is a&lt;br /&gt;potential enzyme employed in the starch processing and&lt;br /&gt;widely used in food industries, but this enzyme is still&lt;br /&gt;imported. The local enzyme production would be more&lt;br /&gt;economist and useful for its broad applications. Here we&lt;br /&gt;report α-amylase from indigenous bacteria TII-12 which was&lt;br /&gt;purified and characterized, as well as analyzed its hydrolysis&lt;br /&gt;product on cassava starch. The enzyme of Bacillus&lt;br /&gt;stearothermophilus TII-12 partially purified by ultrafiltration,&lt;br /&gt;acetone precipitation and gel filtration (Sephadex G-100)&lt;br /&gt;showed the reduced total activity, total protein and yield, but&lt;br /&gt;increased the specific activity. The enzyme had a Km of 1,06&lt;br /&gt;mg/ml and Vmax of 1,21 mol/min, with optimal activity at pH 7&lt;br /&gt;and 90oC. An apparent molecular mass was of 192.932,8&lt;br /&gt;Dalton, as estimated by Native-Polyacrylamide Agarose Gel&lt;br /&gt;electrophoresis. Its activity was inhibited by the divalent&lt;br /&gt;cation chelator such as EDTA and CuSO4 but activated by&lt;br /&gt;calcium ion. Hydrolysis products of this enzyme on cassava&lt;br /&gt;starch were glucose, dextrin, maltose and oligosaccharides.&lt;br /&gt;After 24 hours of hydrolysis, the concentration of glucose&lt;br /&gt;and maltose reached 51.970 and 10.090 ppm, respectively.&lt;br /&gt;The thermostable α-amylase of TII-12 is an endo-α-amylase&lt;br /&gt;and prospective to be applied on starch liquefaction with&lt;br /&gt;high temperature process.&lt;/p&gt;