Thermostable α-amylases hold a very important place in commercial industrial applications in Sri Lanka. Therefore, the main aim of this study was to identify superior Bacillus strain and optimize growth conditions th...Thermostable α-amylases hold a very important place in commercial industrial applications in Sri Lanka. Therefore, the main aim of this study was to identify superior Bacillus strain and optimize growth conditions that could yield high α-amylase production. Three Bacillus strains, B. amyloliquefaciens ATCC 23350, B. licheniformis ATCC 14580 and B. megaterium ATCC 14581 were used for the study. Shake flask culture experiments were conducted to identify the effect of various fermentation conditions such as growth temperature, incubation period, carbon source, nitrogen source, initial pH and carbon concentration on extracellular α-amylase production. DNSA assay was carried out to determine the enzyme activity. The highest temperature for enzyme activity was reported by B. licheniformis at 85°C, followed by B. amyloliquefaciens at 75°C and B. megaterium at 45°C. Both B. amyloliquefaciens and B. licheniformis were able to give their optimum enzyme production at 37°C, while B. megaterium at 30°C in 150 rpm with initial pH of 7. B. licheniformis and B. amyloliquefaciens gave their optimum yield of the enzyme after 48 h of incubation while B. megaterium gave after 24 h of incubation. Among the carbon sources tested cassava starch was able to give the highest enzyme production. For B. amyloliquefaciens, the highest yield of the enzyme was obtained with 2% of starch, tryptone as a nitrogen source and initial pH of 7. Maximum enzyme production for B. licheniformis was obtained with 1.5% of starch, KNO<sub>3</sub> as a nitrogen source and initial pH of 6. For B. megaterium 1% of starch, tryptone and pH 7.5 induced the optimum α-amylase production. According to the results obtained, B. amyloliquefaciens is the highest thermostable alpha amylase producer. However, according to the industrial requirement, B. licheniformis can also be used as an enzyme producer due to its stability in higher temperatures.展开更多
This study describes variation of intron-3 of α-amylase gene from 156 breeds of adzuki beans using SSCP(single-strand conformation polymorphism)analysis. Based on α-amylase gene structure and sequence, A pair of P...This study describes variation of intron-3 of α-amylase gene from 156 breeds of adzuki beans using SSCP(single-strand conformation polymorphism)analysis. Based on α-amylase gene structure and sequence, A pair of PCR primers, F (CCTACATTCTAACACACCCT) and R (GCATATTGTGCCAGTACAAT) were designed to amplify intron-3 fragments of α-amylase gene. 14 variant types were detected, including 13, 9, 10, 4 variant types in the wild, weed, locally cultivated and modern brought-up adzuki beans respectively, 9, 8, 7 variant types of the wild adzuki beans from Japan, China and Korea respectively, and some other variant types in the local adzuki beans from China and Bhutan. 60% of subjects of cultivated races were found to be EE type in the experiment. In addition, sequence analysis of intron-3 of α-amylase gene from 8 variant types reveals the evolution process of various variant types in adzuki beans.展开更多
In the perifused fura-2 loaded exocrine pancreatic acinar cell line AR4-2J pulses of high potassium induced repetitive increases in intracellular calcium. Attached cells when stimulated with high potassium secreted la...In the perifused fura-2 loaded exocrine pancreatic acinar cell line AR4-2J pulses of high potassium induced repetitive increases in intracellular calcium. Attached cells when stimulated with high potassium secreted large amount of amylase. High potassium-induced secretion was dependent both on the concentration of potassium and duration of stimulation. High potassium induced increases in intracellular calcium were inhibited by voltage-dependent calcium channel antagonists with an order of potency as follows: nifedipine > ω-agatoxin IVA > ω-conotoxin GVIA. In contrast, the L-type calcium channel antagonist nifedipine almost completely inhibited potassium-induced amylase secretion, whereas the N-type channel antagonist ω-conotoxin GVIA was without effect. The P-type channel antagonist ω-agatoxin IVA had a small inhibitory effect, but this inhibition was not significant at the level of amylase secretion. In conclusion, the AR4-2J cell line possesses different voltage-dependent calcium channels (L, P,N) with the L-type predominantly involved in depolarization induced amylase secretion.展开更多
In order to improve the thermostability of β- 1,3-1,4-glucanase, evolutionary molecular engineering was used to evolve the β-1,3-1,4-glucanase from Bacillus subtilis ZJF-1A5. The process involves random mutation by ...In order to improve the thermostability of β- 1,3-1,4-glucanase, evolutionary molecular engineering was used to evolve the β-1,3-1,4-glucanase from Bacillus subtilis ZJF-1A5. The process involves random mutation by error-prone PCR and DNA shuffling followed by screening on the filter-based assay. Two mutants, EGsl and EGs2, were found to have four and five amino acid substitutions, respectively. These substitutions resulted in an increase in melting temperature from Tm=62.5℃ for the wild-type enzyme to Tm=65.5℃ for the mutant EGsl and 67.5℃ for the mutant EGs2. However, the two mutated enzymes had opposite approaches to produce reducing sugar from lichenin with either much higher （28%） for the former or much lower （21.6%） for the latter in comparison with their parental enzymes. The results demonstrate that directed evolution is an effective approach to improve the thermostability of a mesophilic enzyme.展开更多
【目的】探讨发芽-挤压膨化-高温α-淀粉酶协同处理对速食糙米粉的冲调性、流动性、色度、风味和淀粉消化性能等品质特性的影响,为高品质速食糙米食品的加工提供理论依据。【方法】糙米经发芽处理后采用高温α-淀粉酶辅助挤压膨化协同处...【目的】探讨发芽-挤压膨化-高温α-淀粉酶协同处理对速食糙米粉的冲调性、流动性、色度、风味和淀粉消化性能等品质特性的影响,为高品质速食糙米食品的加工提供理论依据。【方法】糙米经发芽处理后采用高温α-淀粉酶辅助挤压膨化协同处理(Extrusion-Germinated Brown Rice-Enzyme,EGBRE),以糙米发芽直接挤压膨化处理(Extrusion-Germinated Brown Rice,EGBR)、糙米不发芽分别采用高温α-淀粉酶辅助挤压膨化协同处理(Extrusion-Brown Rice-Enzyme,EBRE)和直接挤压膨化处理(Extrusion-Brown Rice,EBR)3种方式作为对照,分别比较分析糙米粉的水溶性指数、吸水性指数、冲调结块率、分散时间、粘度以及其色度、挥发性物质、淀粉消化性能等品质特性,并进行整体感官评分。【结果】高温α-淀粉酶处理能显著提高糙米粉的溶解性,其中,EGBRE较EGBR水溶性指数提高2.11倍,EBRE较EBR水溶性指数提高2.55倍;而发芽处理对糙米粉的溶解性影响不显著。高温α-淀粉酶处理能显著降低糙米粉冲调后的粘度,其中,EGBRE较EGBR粘度下降60.0%,EBRE较EBR粘度下降31.3%;而发芽处理对糙米糊粘度影响不明显。高温α淀粉酶处理还能显著增加糙米粉中挥发性物质总量,而发芽处理能降低其脂质氧化产物的含量。发芽、挤压膨化和高温α-淀粉酶三者协同处理能显著降低糙米粉的冲调结块率、缩短分散时间,其中,EGBRE较EGBR、EBRE和EBR的冲调结块率分别下降55.4%、74.8%和84.0%,分散时间分别缩短27.2%、17.3%和52.5%。同时,发芽-挤压-淀粉酶协同处理能显著影响糙米粉的休止角,改善粉体流动性能,但会适当提高粉体的色度。此外,发芽-挤压膨化-高温α-淀粉酶协同处理较直接挤压膨化处理,糙米粉中快消化淀粉比例显著提高,抗性淀粉比例显著降低。【结论】发芽-挤压-淀粉酶协同处理可以显著改善速食糙米粉的品质特征与冲调性,降低结块率,缩短分散时间,降低冲调粘度,提高淀粉的消化性能,适当增加挥发性风味物质含量。展开更多
通过采用匀浆、硫酸铵盐析、透析、DEAE-52阴离子交换层析、Sephacryl S-300凝胶层析等方法从海参体壁中分离纯化并得到了α-1,4淀粉酶。结果表明,该酶具有3条亚基链,分子质量约为420 k D;最适p H值为9.0,最适温度为80℃,90℃条件下保温...通过采用匀浆、硫酸铵盐析、透析、DEAE-52阴离子交换层析、Sephacryl S-300凝胶层析等方法从海参体壁中分离纯化并得到了α-1,4淀粉酶。结果表明,该酶具有3条亚基链,分子质量约为420 k D;最适p H值为9.0,最适温度为80℃,90℃条件下保温30 min后仍有38%以上的相对酶活性,因此,该酶是一种具有较高热稳定性的碱性高温淀粉酶。K+、Fe3+和Mn2+对其有较强的抑制作用,Cu2+和Ca2+对其有较强的激活作用。展开更多
文摘Thermostable α-amylases hold a very important place in commercial industrial applications in Sri Lanka. Therefore, the main aim of this study was to identify superior Bacillus strain and optimize growth conditions that could yield high α-amylase production. Three Bacillus strains, B. amyloliquefaciens ATCC 23350, B. licheniformis ATCC 14580 and B. megaterium ATCC 14581 were used for the study. Shake flask culture experiments were conducted to identify the effect of various fermentation conditions such as growth temperature, incubation period, carbon source, nitrogen source, initial pH and carbon concentration on extracellular α-amylase production. DNSA assay was carried out to determine the enzyme activity. The highest temperature for enzyme activity was reported by B. licheniformis at 85°C, followed by B. amyloliquefaciens at 75°C and B. megaterium at 45°C. Both B. amyloliquefaciens and B. licheniformis were able to give their optimum enzyme production at 37°C, while B. megaterium at 30°C in 150 rpm with initial pH of 7. B. licheniformis and B. amyloliquefaciens gave their optimum yield of the enzyme after 48 h of incubation while B. megaterium gave after 24 h of incubation. Among the carbon sources tested cassava starch was able to give the highest enzyme production. For B. amyloliquefaciens, the highest yield of the enzyme was obtained with 2% of starch, tryptone as a nitrogen source and initial pH of 7. Maximum enzyme production for B. licheniformis was obtained with 1.5% of starch, KNO<sub>3</sub> as a nitrogen source and initial pH of 6. For B. megaterium 1% of starch, tryptone and pH 7.5 induced the optimum α-amylase production. According to the results obtained, B. amyloliquefaciens is the highest thermostable alpha amylase producer. However, according to the industrial requirement, B. licheniformis can also be used as an enzyme producer due to its stability in higher temperatures.
文摘This study describes variation of intron-3 of α-amylase gene from 156 breeds of adzuki beans using SSCP(single-strand conformation polymorphism)analysis. Based on α-amylase gene structure and sequence, A pair of PCR primers, F (CCTACATTCTAACACACCCT) and R (GCATATTGTGCCAGTACAAT) were designed to amplify intron-3 fragments of α-amylase gene. 14 variant types were detected, including 13, 9, 10, 4 variant types in the wild, weed, locally cultivated and modern brought-up adzuki beans respectively, 9, 8, 7 variant types of the wild adzuki beans from Japan, China and Korea respectively, and some other variant types in the local adzuki beans from China and Bhutan. 60% of subjects of cultivated races were found to be EE type in the experiment. In addition, sequence analysis of intron-3 of α-amylase gene from 8 variant types reveals the evolution process of various variant types in adzuki beans.
文摘In the perifused fura-2 loaded exocrine pancreatic acinar cell line AR4-2J pulses of high potassium induced repetitive increases in intracellular calcium. Attached cells when stimulated with high potassium secreted large amount of amylase. High potassium-induced secretion was dependent both on the concentration of potassium and duration of stimulation. High potassium induced increases in intracellular calcium were inhibited by voltage-dependent calcium channel antagonists with an order of potency as follows: nifedipine > ω-agatoxin IVA > ω-conotoxin GVIA. In contrast, the L-type calcium channel antagonist nifedipine almost completely inhibited potassium-induced amylase secretion, whereas the N-type channel antagonist ω-conotoxin GVIA was without effect. The P-type channel antagonist ω-agatoxin IVA had a small inhibitory effect, but this inhibition was not significant at the level of amylase secretion. In conclusion, the AR4-2J cell line possesses different voltage-dependent calcium channels (L, P,N) with the L-type predominantly involved in depolarization induced amylase secretion.
基金Project supported by the National Natural Science Foundation of China (No. 20276064) and Natural Science Foundation of ZhejiangProvince (No. Z304076), China
文摘In order to improve the thermostability of β- 1,3-1,4-glucanase, evolutionary molecular engineering was used to evolve the β-1,3-1,4-glucanase from Bacillus subtilis ZJF-1A5. The process involves random mutation by error-prone PCR and DNA shuffling followed by screening on the filter-based assay. Two mutants, EGsl and EGs2, were found to have four and five amino acid substitutions, respectively. These substitutions resulted in an increase in melting temperature from Tm=62.5℃ for the wild-type enzyme to Tm=65.5℃ for the mutant EGsl and 67.5℃ for the mutant EGs2. However, the two mutated enzymes had opposite approaches to produce reducing sugar from lichenin with either much higher （28%） for the former or much lower （21.6%） for the latter in comparison with their parental enzymes. The results demonstrate that directed evolution is an effective approach to improve the thermostability of a mesophilic enzyme.
文摘【目的】探讨发芽-挤压膨化-高温α-淀粉酶协同处理对速食糙米粉的冲调性、流动性、色度、风味和淀粉消化性能等品质特性的影响,为高品质速食糙米食品的加工提供理论依据。【方法】糙米经发芽处理后采用高温α-淀粉酶辅助挤压膨化协同处理(Extrusion-Germinated Brown Rice-Enzyme,EGBRE),以糙米发芽直接挤压膨化处理(Extrusion-Germinated Brown Rice,EGBR)、糙米不发芽分别采用高温α-淀粉酶辅助挤压膨化协同处理(Extrusion-Brown Rice-Enzyme,EBRE)和直接挤压膨化处理(Extrusion-Brown Rice,EBR)3种方式作为对照,分别比较分析糙米粉的水溶性指数、吸水性指数、冲调结块率、分散时间、粘度以及其色度、挥发性物质、淀粉消化性能等品质特性,并进行整体感官评分。【结果】高温α-淀粉酶处理能显著提高糙米粉的溶解性,其中,EGBRE较EGBR水溶性指数提高2.11倍,EBRE较EBR水溶性指数提高2.55倍;而发芽处理对糙米粉的溶解性影响不显著。高温α-淀粉酶处理能显著降低糙米粉冲调后的粘度,其中,EGBRE较EGBR粘度下降60.0%,EBRE较EBR粘度下降31.3%;而发芽处理对糙米糊粘度影响不明显。高温α淀粉酶处理还能显著增加糙米粉中挥发性物质总量,而发芽处理能降低其脂质氧化产物的含量。发芽、挤压膨化和高温α-淀粉酶三者协同处理能显著降低糙米粉的冲调结块率、缩短分散时间,其中,EGBRE较EGBR、EBRE和EBR的冲调结块率分别下降55.4%、74.8%和84.0%,分散时间分别缩短27.2%、17.3%和52.5%。同时,发芽-挤压-淀粉酶协同处理能显著影响糙米粉的休止角,改善粉体流动性能,但会适当提高粉体的色度。此外,发芽-挤压膨化-高温α-淀粉酶协同处理较直接挤压膨化处理,糙米粉中快消化淀粉比例显著提高,抗性淀粉比例显著降低。【结论】发芽-挤压-淀粉酶协同处理可以显著改善速食糙米粉的品质特征与冲调性,降低结块率,缩短分散时间,降低冲调粘度,提高淀粉的消化性能,适当增加挥发性风味物质含量。
