• Microbiology and Biotechnology Letters
• /
• v.49 no.1
• /
• pp.45-56
• /
• 2021

Improved amylases were developed from protoplast fusants of two amylase-producing Aspergillus species. Twenty regenerated fusants were screened for amylase production using Remazol Brilliant Blue agar. Crude enzyme extracts produced by solid state fermentation of rice bran were assayed for activity. Three variable factors (temperature, pH and enzyme type) were optimized to increase the amylase activity of the parents and selected fusants using rice bran medium and solid state fermentation. Analysis of this optimization was completed using the Central Composite Design (CCD) of the Response Surface Methodology (RSM). Amylase activity assays conducted at room temperature and 80℃ demonstrated that Aspergillus designates, T5 (920.21 U/ml, 966.67 U/ml), T13 (430 U/ml, 1011.11 U/ml) and T14 (500.63 U/ml, 1012.00 U/ml) all exhibited improved function making them the preferred fusants. Amylases produced from these fusants were observed to be active over the entire pH range evaluated in this study. Fusants T5 and T14 demonstrated optimal activity under acidic and alkaline conditions, respectively. Fusants T13 and T14 produced the most amylase at 72 h while parents TA, TC and fusant T5 produced the most amylase after 96 h of incubation. Response surface methodology examinations revealed that the enzyme from fusant T5 was the optimal enzyme demonstrating the highest activity (1055.17 U/ml) at pH 4 and a temperature of 40℃. This enzyme lost activity with further increases in temperature. Starch hydrolysis using fusant T5 gave the highest yield of glucose (1.6158 g/100 ml). The significant activities of the selected fusants at 28 ± 2℃ and 80℃ and the higher sugar yields from cassava starch hydrolysis over their parental strains indicate that it is possible to improve amylase activity using the protoplast fusion technique.

• Journal of Applied Biological Chemistry
• /
• v.49 no.4
• /
• pp.135-139
• /
• 2006

A alkalophilic strain, Bacillus licheniformis MH31 producing an alkaline protease was isolated from mine soil of Boryeong in Korea. Production of a high level of alkaline protease was achieved 42 h after incubation when the bacterium was grown at pH 9.0 and $35^{\circ}C$ in Horikoshi medium supplemented with 0.5%(w/v) starch and 1%(w/v) skim milk as carbon and nitrogen source, respectively. The molecular weight of partially purified enzyme was estimated to be 30 kDa by SDS-PAGE and its optimum pH was pH 10. The enzyme showed optimum temperature at $50^{\circ}C$, and was stable up to $60^{\circ}C$ after 1 h incubation. The protease was strongly inhibited by 1 mM of PMSF which was known well as strong inhibitor of serine proteases, but almost not inhibited by 5 mM of EDTA and 1,10-phenanthroline. When the protein hydrolysis products of 1% skim milk by partially purified protease was compared with available commercial proteases using HPLC analysis, most of hydrolysis products were detected below molecular weight of 10,000 and the hydrolysis ratio of purified enzyme was 24.8% lower than those(above 32%) of commercial proteases.

• Journal of Applied Biological Chemistry
• /
• v.43 no.4
• /
• pp.240-245
• /
• 2000

Two genes encoding maltooligosyltrehalose synthase (SaMTS) and maltooligosyltrehalose trehalohydrolase (SaMTH) were isolated from a hyperthermophilic microorganism, Sulfolobus acidocaldarius (ATCC 49462). ORFs of the SaMTS and SaMTH genes are 2,163 and 1,671 bp long and encode 720 and 556 amino acid residues, respectively. A bifunctional fusion enzyme (SaMTSH) was constructed through the gene fusion of SaMTS and SaMTH. Recombinant SaMTS, SaMTH, and SaMTSH fusion enzyme were overexpressed in E. coli BL21. SaMTS and SaMTH produced trehalose and maltotriose from maltopentaose in a sequential reaction. SaMTSH fusion enzyme catalyzed the sequential reaction in which the formation of maltotriosyltrehalose was followed by hydrolysis leading to the synthesis of trehalose and maltotriose. The SaMTSH fusion enzyme showed the highest activity at pH 5.0-5.5 and $70-75^{\circ}C$. SaMTS, SaMTH, and SaMTSH fusion enzyme were active in soluble starch, which resulted in the production of trehalose.

• Microbiology and Biotechnology Letters
• /
• v.9 no.4
• /
• pp.185-190
• /
• 1981

1. $\alpha$-amylase from B. circulans F-2 was purified with specific activity 55.0 u/mg. protein (about 23 times of the original specific activity) and the yield of 25.5%, by means of corn starch absorption, salting out with ammonium sulfate (80% saturation), gel filtration on Bio-Gel P-100 and DE-32 column chromatography. 2 The purified enzyme showed two closely migrated protin bands on polyacrylamide disc gel electrophoresis, both of which have amylase activity judging from the activity staining of the gel. On SDS-polyacrylamide disc gel electrophoresis, however, the purified enzyme showed a single band suggesting that those two bands are the charge isomers of an amlyase having the slightly different charge. 3. Plot of log mobility of two bands versus polyacrylamide gel concentration according to Hedrick and Smith gave the parallel lines indicating them to be charge isomers. 4. To confirm the action pattern of two enzyme protein bands, each band was separated and was eluted from the gel and eluates were incubated with soluble starch. Oligosaccharide pattern produced by each eluate was examined by paper chromatography. The eluates of two bands showed the same action pattern. 5. The maltohexaose was the only hydrolysis product of soluble starch in the early stage of hydrolysis.

• KSBB Journal
• /
• v.9 no.5
• /
• pp.499-505
• /
• 1994

Bacillus macerans cyclodextrin glucanotransferase(EC 2.4.1.19: 1, 4-${\alpha}$-D(1, 4-${\alpha}$-glucano)-transferase, CGTase) was purified by the technique of starch adsorption and DEAE-cellulose column chromatography. The molecular weight of the enzyme was 67,000, consisting of a subunit. The enzyme converted starch into ${\alpha}$-, ${\beta}$-, and ${\gamma}$-CD in the relative amounts of 1:1.68:0.32, respectively. In the early reaction period, maltohexose was formed mainly by the coupling reaction of ${\alpha}$-CD with D-glucose and then other oligosaccharides. Maltotetrose was formed mainly from ${\alpha}$-CD in the initial stage of hydrolysis of the enzyme and then small amount of other oligosaccharides. Maltotriose was a good substrate for the enzyme and maltosyl or D-glucopyranosyl group can be transfered from this sugar. In this work, D-glutosyl transfer was premiered.

• Applied Biological Chemistry
• /
• v.40 no.6
• /
• pp.508-513
• /
• 1997

Thermal characteristics and granular morphology on enzyme-resistant starches (RS) formed during heat-moisture treatment (HMT) and retrogradation were investigated in high amylose corn starches, Hylon V and Hylon VII. With each treatment, both starches showed a similar trend in the increase of RS, but RS yield of Hylon VII is higher than that of Hylon V. Specially, RS was increased remarkably by HMT. It was more than doubled from 11.4% to 26.6% for Hylon V and from 15.9% to 32.8% for Hylon VII. A small increase of RS resulted from retrogradation. HMT on starch increased gelatinization temperature, decreased enthalpy. Retrograded starch exhibited small three endothermic transitions at $94^{\circ}C$, $110^{\circ}C$ and $140^{\circ}C$ in differential scanning calorimetry (DSC) thermogram due to the remained ungelatinized starch granules, dissociation of amylose-lipid complex and melting of recrystallized amylose, respectively. Enzyme-resistant starches isolated from native and heat-moisture treated starches showed a broad endothermic transition at higher temperature than native starch, while retrograded starch exhibited a very sharp peak at ${\sim}150^{\circ}C$ due to the melting of amylose crystallites. Under microscopy, starch granules with HMT was not changed, but retrograded starches showed the aggregates of starch granules because amylose leached out during gelatinization. Iodine stained RS clearly showed the differences in enzyme hydrolysis on the native, heat-moisture treated and retrograded starches.

• Asian-Australasian Journal of Animal Sciences
• /
• v.30 no.2
• /
• pp.171-180
• /
• 2017

Objective: This study investigated the association of enzyme-producing microbes and their enzymes with starch and hemicellulose degradation during fermentation of total mixed ration (TMR) silage. Methods: The TMRs were prepared with soybean curd residue, alfalfa hay (ATMR) or Leymus chinensis hay (LTMR), corn meal, soybean meal, vitamin-mineral supplements, and salt at a ratio of 25:40:30:4:0.5:0.5 on a dry matter basis. Laboratory-scale bag silos were randomly opened after 1, 3, 7, 14, 28, and 56 days of ensiling and subjected to analyses of fermentation quality, carbohydrates loss, microbial amylase and hemicellulase activities, succession of dominant amylolytic or hemicellulolytic microbes, and their microbial and enzymatic properties. Results: Both ATMR and LTMR silages were well preserved, with low pH and high lactic acid concentrations. In addition to the substantial loss of water soluble carbohydrates, loss of starch and hemicellulose was also observed in both TMR silages with prolonged ensiling. The microbial amylase activity remained detectable throughout the ensiling in both TMR silages, whereas the microbial hemicellulase activity progressively decreased until it was inactive at day 14 post-ensiling in both TMR silages. During the early stage of fermentation, the main amylase-producing microbes were Bacillus amyloliquefaciens (B. amyloliquefaciens), B. cereus, B. licheniformis, and B. subtilis in ATMR silage and B. flexus, B. licheniformis, and Paenibacillus xylanexedens (P. xylanexedens) in LTMR silage, whereas Enterococcus faecium was closely associated with starch hydrolysis at the later stage of fermentation in both TMR silages. B. amyloliquefaciens, B. licheniformis, and B. subtilis and B. licheniformis, B. pumilus, and P. xylanexedens were the main source of microbial hemicellulase during the early stage of fermentation in ATMR and LTMR silages, respectively. Conclusion: The microbial amylase contributes to starch hydrolysis during the ensiling process in both TMR silages, whereas the microbial hemicellulase participates in the hemicellulose degradation only at the early stage of ensiling.

• Food Science and Biotechnology
• /
• v.14 no.5
• /
• pp.681-684
• /
• 2005

A maltopentaose-producing amylase (G5-amylase) from Bacillus megaterium KSM B-404 was applied to retard bread retrogradation. Retrogradation rates were determined by differential scanning calorimetry. Gel permeation chromatography determined changes in maltooligosaccharide composition and the molecular weight profiles of carbohydrate tractions. The baking process produced maltopentaose and maltotriose by the hydrolysis of starch molecules into small units. Amylose and amylopectin degradation as well as maltooligosaccharides produced by the enzyme were likely responsible for retarding starch retrogradation. Overall, addition of G5-amylase reduced the starch retrogradation rate, and was as effective as Novamyl(R), a commercial enzyme.

• Microbiology and Biotechnology Letters
• /
• v.18 no.3
• /
• pp.251-259
• /
• 1990

Asp. usumii IAM 2185 was selected as a strain producing the powerful raw starch digesting glucoamylase. The optimum initial pH, the optimum temperature and the optimum cultural time for the enzyme production on wheat bran medium were pH 6-8,25-$30^{\circ}C$ and 72 hrs, respectively. The addition of ammonium nitrate and albumin on wheat bran medium, respectively, increase slightly the enzyme production. The enzyme was purified by ammonium sulfate fractionation, CM-cellulose and DEAE-cellulose column chromatography. The specific activity of the purified enzyme was 34.3 U/mg protein and the yield of enzyme activity was 10.3%. The purified enzyme showed a single band on polyacrylamide disc gel electrophoresis and its molecular weight was estimated to be 67,000 by SDS polyacrylamide disc gel electrophoresis. The isoelectric point for the purified enzyme was pR 3.7. The optimum temperature and optimum pH were $60^{\circ}C$and pH 3.0 and the purified enzyme was stable in the pH range of 1.0-11.0. The purified enzyme was stable below $50^{\circ}C$ and its thermostability was greatly increased by the addition of $Ca^{2+}$. The purified enzyme showed a high hydrolysis rate on various raw starches such as corn, rice, yam, arrow root, sweet potato and glutinous rice.

• Microbiology and Biotechnology Letters
• /
• v.17 no.4
• /
• pp.349-357
• /
• 1989

Uncooked starch can be effectively saccharified in an enzyme reaction system containing attrition-milling media. To develope the high efficiency bioattritor, an agitated bead type bioreactor was constructed, and its effectiveness was evaluated. The optimal operation condition of bioattritor was found to be 300 g glass bead/L, 200 rpm, standard type impeller for 220 g/L of uncooked corn starch. The torque under the various operational conditions were also measured. The interrelation-ship between energy consumption for agitation of attrition-milling media and enhanced extent of saccharification of uncooked starch was evaluated, Power consumption was measured to be around 1.53 watt/L under the optimal operation condition. The attrition coupled enzyme reaction system is identified to tie a very excellent energy saying process for saccharification of uncooked starch, and seems to have a bright prospect of industrial application.