α-Glucosidase inhibitory activity by the flower buds of Lonicera japonica Thunb

In the present study, the methanol extract from the flower buds of Lonicera japonica Thunb. was investigated on rat intestinal α-glucosidase inhibitory activity. This extract enriched in polyphenols, showed a potent α-glucosidase inhibitory activity with maltose as a substrate in enzyme assay. The extract was then applied to MCI-gel column chromatography with a water–methanol gradient to give three fractions: fraction 1 (F-1, water), fraction 2 (F-2, 80% methanol in water), and fraction 3 (F-3, 100% methanol). F-2 showed the highest maltase inhibitory activity compared with F-1 and F-3, and down-regulated α-glucosidase activity for the maltose hydrolysis in Caco-2 monolayer. In maltose-loaded SD-rats, F-2 significantly reduced postprandial blood glucose level. 3,5-Dicaffeoylquinic acid was found to be the potent maltase inhibitor, whereas chlorogenic acid and rutin showed a weak inhibitory activity against maltase. This is the first report on the mammalian α-glucosdiase inhibition of L. japonica. The result presented here suggests a use of the extract of L. japonica for antidiabetes.     

α-GLUCOSIDASE ACTIVITY OF SORGHUM AND BARLEY MALTS

Sorghum malt α-glucosidase activity was highest at pH 3.75 while that of barley malt was highest at pH 4.6. At pH 5.4 employed in mashing sorghum malt α-glucosidase was more active than the corresponding enzyme of barley malt. α-Glucosidase was partly extracted in water but was readily extracted when L-cysteine was included in the extraction buffer, pH 8. Sorghum malt made at 30°C had higher α-glucosidase activities than the corresponding malts made at 20°C and 25°C. Nevertheless, the sorghum malts made at 20°C and 25°C produced worts which contained more glucose than worts of malt made at 30°C. Although barley malts contained more α-glucosidase activity than sorghum malts, the worts of barley had the lowest levels of glucose. The limitation to maltose production in sorghum worts, produced at 65°C, is due to inadequate gelatinization of starch and not to limitation to β-amylase and α-amylase activities. Gelatinization of the starch granules of sorghum malt in the decantation mashing procedure resulted in the production of sorghum worts which contained high levels of maltose, especially when sorghum malt was produced at 30°C. Although the β-amylase and α-amylase levels of barley malt was significantly higher than those of sorghum malted optimally at 30°C, sorghum worts contained higher levels of glucose and equivalent levels of maltose to those of barley malt. It would appear that the individual activities of α-glucosidase, α-amylase and β-amylase of sorghum malts or barley malts do not correlate with the sugar profile of the corresponding worts. In consequence, specifications for enzymes such as α-amylase and β-amylase in malt is best set at a range of values rather than as single values.     

New source of the thermostable α-glucosidase suitable for single step starch processing

Thermus thermophilus contains α-glucosidase that is located in the cell cytoplasm. The highest enzyme activity was achieved after 25–30 h of microorganism cultivation at 70 °C in a medium containing 0.8% peptone, 0.4% yeast extract and 0.2% Na Cl. Addition of 2% of starch, maltose or glucose to the medium enhanced the enzyme activity by 83, 72 and 31%, respectively. The enzyme was purified 7.2-fold by combination of ammonium sulphate precipitation and ion-exchange chromatography on Sepharose CL-6B. The enzyme preparations exhibited highest specific activity for pNPG hydrolysis (1.94 U mg⁻¹) at pH 6.2 and 85 °C. The activity of partially purified enzyme was almost unchanged during 5 h of incubation at 75 °C in phosphate-citrate buffer (pH 6.2) and the half-life of this enzyme incubated at 85 °C was 2 h. Rate of pNPG cleavage catalysed by the α-glucosidase was 9.5-times higher than that in the case of maltose hydrolysis. Furthermore, this enzyme shows remarkable activity toward maltose and maltotriose. With maltotetraose, maltopentaose and maltohexaose the reaction rate decreased with increase in molecular weight of the substrate.     

Enhanced antioxidant and antidiabetic activities of barley and wheat after soaking with tea catechin

Barley and wheat were investigated as natural α-glucosidase inhibitors having potent inhibitory activities against the antidiabetic enzymes α-glucosidase, maltase, and sucrase. After soaking with tea catechin as an elicitor, extracts of barley and wheat were prepared using thermal treatment in an autoclave at 121°C for 15 min, then the in vitro antioxidant and antidiabetic activities were determined. The total soluble phenolics contents of barley and wheat during soaking were increased by addition of tea catechin, which also contributed to the peroxyl radical-scavenging activity and the reducing capacity. The rat α-glucosidase, maltase, and sucrase inhibitory activities of barley were increased with an increase in the added tea catechin concentration from 0.1% to 0.5%. Tea catechin may have a role as an elicitor for production of phenolics that exhibit antioxidant activities and rat-intestinal maltase and sucrase inhibitory activities during the soaking process of barley and wheat.     

海栖热袍菌α-葡萄糖苷酶基因aglA的克隆、表达及酶学性质

主要研究了在大肠杆菌中克隆和表达海栖热袍菌(Thermotoga maritima)的一个α-葡萄糖苷酶(TM1834).通过PCR方法克隆编码T.maritima的一个α-葡萄糖苷酶基因aglA,构建重组质粒pHsh-AglA,电击转化Escherichia coli JM109,通过热激诱导高效表达.SDS-PAGE检测出蛋白相对分子质量约55 000,经热处理,阴离子交换层析和疏水层析纯化后的α-葡萄糖苷酶最适反应温度为90℃,最适反应pH为7.5,在pH 6.5～8.5,温度65～100℃之间酶活仍达到50％以上.在辅助因子NAD+,Mn2+和还原剂DTT的存在条件下达到最高酶活.     

响应面法优化微波辅助酶解制备α-葡萄糖苷酶抑制活性肽工艺

为了优化微波辅助酶解制备α-葡萄糖苷酶抑制活性肽工艺,以冷榨花生蛋白粉为原料,以酶解得到的α-葡萄糖苷酶抑制活性肽复合物对α-葡萄糖苷酶的抑制率为考察指标,在单因素实验基础上,通过响应面Box-Benhnken实验设计进行工艺优化。结果表明,最优工艺条件为底物浓度9.77%、加酶量0.94%、温度59 ℃、时间10 min、pH9.0、微波功率1000 W;此工艺条件下的α-葡萄糖苷酶抑制活性肽复合物对α-葡萄糖苷酶的抑制率的响应面模型预测值为84.80%,验证实验的抑制率为90.21%±0.93%,两者的差异值为6.38%。本研究结果为花生α-葡萄糖苷酶抑制活性肽的分离、纯化和应用等研究提供了理论基础。     

新型碱性低分子量β-葡萄糖苷酶基因的分离与酶学性质研究

目的:获得新型β-葡萄糖苷酶编码基因,并对其表达产物进行酶学性质研究。方法:采用宏基因组学方法提取兔盲肠内微生物总DNA,构建DNA文库,通过筛选培养基获得产β-葡萄糖苷酶的克隆,测序获得其插入基因序列,氨基酸多重序列比对分析其序列特征,并用DNS显色法测定表达产物在不同条件下的酶活力。结果:在文库中获得一个基因片段nglu07,能编码一个低分子量的β-葡萄糖苷酶。nglu07基因片段长180bp,编码60个氨基酸残基。与已提交的糖苷水解酶Ⅰ家族成员进行氨基酸多重序列比对表明nglu07具有预测的活性位点Glu4与底物结合位点Tyr47,其最适反应温度为50℃,最适pH为10.0,粗酶活为8.12U/mL。结论:成功获得一新型低分子量的碱性β-葡萄糖苷酶编码基因,其蛋白温度稳定性高,在pH4.0～11.0的环境中均能保持较高的酶活力,具有作为食品工业用酶以及碱性酶的潜力,尤其在食品饮料加工、棉织品生物整理、洗涤及废纸脱墨等工业方面具有一定的应用价值。     

ASSESSMENT OF BACILLUS LICHENIFORMIS α-AMYLASE AS A CANDIDATE ENZYME FOR GENETIC ENGINEERING OF MALTING BARLEY1

Bacillus licheniformis α-amylase, a thermostable starch-degrading enzyme, has been assessed as a candidate enzyme for the genetic transformation of malting barley. The temperature optimum, pH optimum and thermostability of B. licheniformis α-amylase were compared with those of barley α-amylase. The bacterial enzyme has a higher pH optimum (?9), a higher temperature optimum (?90°C) and much higher thermostability at elevated temperatures than the barley enzyme. The specific activity of the bacterial enzyme under conditions of pH and temperature relevant to the brewing process (pH 5.5, 65°C) is ?1.5-fold higher than that of the barley enzyme. Measurements of α-amylase activity during a micro-mash showed that the bacterial enzyme is at least as stable as the barley enzyme under these conditions, and that a level of expression for the bacterial enzyme corresponding to ?0.5% of total malt protein would approximately double the α-amylase activity in the mash. B. licheniformis α-amylase activity was rapidly eliminated by boiling following mashing as would occur during brewing. The combined results suggest that barley expressing the bacterial enzyme may be useful in the brewing process.     

EXTRACTION OF α-GLUCOSIDASE FROM GERMINATED BARLEY KERNELS

α-Glucosidase activities were extracted from flour of lyophilized, germinated barley seeds. To establish conditions resulting in maximal extraction of α-glucosidase activity from flour of germinated barley, we tested different buffer pH's, extraction times and temperatures, and additions of sulfhydryl reducing reagents, salts, and detergents to the extraction buffer. More enzyme activity was extracted as the extraction buffer pH increased from 3 to 9. The sulfhydryl reagents studied did not significantly increase extractable activity. The extractable activity rose with increased NaCl concentrations up to 2 M. Of the six detergents tested, the zwitterionic detergent CHAPS was most effective in solubilizing α-glucosidase activity, and when combined with NaCl resulted in a doubling in extractable activity as compared with phosphate buffer at pH 8.0. Extraction time and temperature were not critical for the actual solubilization of activity but temperature was quite critical in retaining solubilized α-glucosidase activity in crude extracts.     

酶解丝素蛋白制备ɑ-葡萄糖苷酶抑制肽的研究

探讨利用碱性蛋白酶酶解丝素蛋白制备ɑ-葡萄糖苷酶抑制肽的工艺条件。以丝素肽对ɑ-葡萄糖苷酶的体外抑制活性和水解度(DH)为主要评价指标,通过正交试验优化了ɑ-葡萄糖苷酶抑制肽的工艺条件。结果表明,在最佳工艺条件为温度60℃、反应pH8.5、底物质量浓度5%、加酶量为1200 U/g、反应时间100 min下测得丝素肽对ɑ-葡萄糖苷酶具有较高抑制率,为44.90%,此时DH为16.80%。其抑制活性与浓度有较大的依赖关系,而作用时间控制在40 min即可。此外,通过对丝素蛋白肽蔗糖和葡萄糖含量的检测,结果表明,尽管丝素肽中含有微量的葡萄糖,但抑制干扰作用甚微。     

Purification and characterisation of α-glucosidase inhibitory peptides from defatted camellia seed cake

Enzymatically derived peptides from different proteins have displayed the potential to provide health benefits. Present research aims to obtain the functional peptides and improve the economic value of camellia seed cake via enzymatic hydrolysis. Camellia seed cake protein hydrolysates (CSCPH) prepared with alcalase demonstrated higher α-glucosidase inhibitory activity. After ultrafiltration, the α-glucosidase inhibitory activity of different components was compared. The component (CSCPH-IV) with the highest activity was selected for further separation and purification. The peptides were purified using reverse-phase high-performance liquid chromatography (RP-HPLC) twice and identified as GHSLESIK, GLTSLDRYK and SPGYYDGR by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Subsequently, the preliminarily inhibitory effect of these peptides on α-glucosidase activity was demonstrated by the molecular docking model. These results showed that three novel peptides isolated from the hydrolysates of camellia seed cake have the potential to be used as anti-diabetic compounds for the development of functional foods.     

Characterization of a β-glucosidase isolated from an alpeorujo strain of Candida adriatica

A β-glucosidase-producing strain, Candida adriatica CECT13142, was isolated from olive oil wastes (alpeorujo) and identified by PCR/restriction fragment length polymorphism of the rDNA internal transcribed spacer and sequence analysis of the D1/D2 region of the 26S rRNA gene techniques. The enzyme was purified by sequential chromatography on DEAE-cellulose and Sephadex G-100. The relative molecular mass of the enzyme was estimated to be 50 kDa by SDS-PAGE. The hydrolytic activity of the β-glucosidase had an optimum pH of 8.2 and an optimum temperature of 40°C. The enzyme displayed high substrate specificity and high catalytic efficiency (K_m 0.85 mM, V_max 12.5 U/g of cells) for p-nitrophenyl-β-D-glucopyranoside. Although β-glucosidases have been purified and characterized from several other organisms, the C. adriatica β-glucosidase is able to have optimal activity at alkaline pH.     

产β-葡萄糖苷酶野生真菌的筛选鉴定及酶学性质研究

从原始森林腐木的土壤中筛选得到1株高产β-葡萄糖苷酶活力菌株Lcxs9,表型分析及ITS rDNA序列分子鉴定为芽枝霉菌,酶活为7.18U/mL,在pH值为4~9范围内,60℃以下酶活力稳定,酶谱分析芽枝霉菌Lcxs9可以产2种β-葡萄糖苷酶。该文首次报道芽枝霉菌产高活性β-葡萄糖苷酶。     

THE BARLEY PROTEIN DEGRADATION: EFFECT OF NEUTRAL PROTEINASE CONCENTRATION ON PROTEIN DEGRADATION KINETICS

The time course of barley protein degradation was investigated in two cases: (i) in mashing of raw barley with different concentrations of bacterial neutral proteinase in the presence of α-amylase. and (ii) in mashing of spent grains, remaining after barley syrup preparation, with different proteinase concentrations. Protein solubilisation and α-amino nitrogen liberation rates decrease in the course of barley and spent grains hydrolysis. Initial protein degradation rates in spent grains mashing are lower than in native barley. Gel-chromatography of supernatants of the mash in the course of hydrolysis has indicated that the proteinase becomes increasingly occupied with solubilised proteins as the process proceeds. plotting the kinetics data for protein solubilisation in Foster-Niemann coordinates straight lines with high correlation (r=0·999) for both cases of hydrolysis have been obtained. For different enzyme concentrations the straight lines have been closely parallel with positive slope. A high correlation between ordinate intercept in Foster-Niemann coordinates and proteinase concentrations has been obtained. Linear correlations in Foster-Niemann coordinates for α-amino nitrogen liberation process have also been observed. However, for α-amino nitrogen liberation, the correlation between the ordinate intercept and proteinase concentration has not been achieved. The possibilities of application of kinetics parameters (ordinate intercept slope) as quantitative measures of enzyme efficiency in protein degradation under different conditions has been emphasised.     

普鲁兰酶协同α-葡萄糖苷酶降低青稞快消化淀粉含量酶解工艺优化

以青稞粉为原料,通过普鲁兰酶协同α-葡萄糖苷酶降低青稞快消化淀粉（RDS）含量。通过单因素试验和响应面试验确定降低青稞快消化淀粉含量的最优酶解工艺条件,并测定α-葡萄糖苷酶的抑制率评价其体外降糖活性。结果表明,最佳酶解工艺条件为：普鲁兰酶添加量200 U/g、α-葡萄糖苷酶添加量80 U/g、料液比1∶15(g∶mL)、酶解时间3 h、酶解温度55℃。在此优化条件下,青稞粉快消化淀粉含量为54.95%,比未处理过的青稞快消化淀粉含量降低了20.22%。体外降糖活性测定结果表明,与原粉相比,酶解粉的α-淀粉酶和α-葡萄糖苷酶的抑制率分别增加了68.1%和50.4%,表明经过双酶协同酶解后,青稞淀粉的体外降血糖活性明显提高。     

不同干酪乳杆菌菌株对α-葡萄糖苷酶抑制作用的比较

从14株干酪乳杆菌中筛选出11株具有凝乳作用的菌株,采用以麦芽糖酶为靶向酶的α-葡萄糖苷酶抑制剂体外筛选模型,分析了不同干酪乳杆菌发酵脱脂乳上清对α-葡萄糖苷酶的抑制作用。结果表明,不同菌株发酵脱脂乳上清对麦芽糖酶的抑制活性具有明显的菌株特异性,且随着发酵时间延长,α-葡萄糖苷酶抑制活性呈现上升趋势。其中,干酪乳杆菌LC2W在37℃发酵10%(w/w)脱脂乳96 h后所得的上清液对麦芽糖酶抑制活性最高,达37.36%,明显高于其它被测试的干酪乳杆菌菌株,表明该菌株具有潜在的抗高血糖作用。     

响应面法优化辣木籽降糖肽的酶法制备工艺及其体外活性评价

为提升辣木籽资源利用度,采用酶法制备辣木籽降糖肽。以蛋白水解度和α-葡萄糖苷酶抑制率为指标,通过单因素实验筛选和响应面法优化最佳的酶解时间、液料比、酶解pH、酶添加量和酶解温度,通过超滤粗分离酶解液制备分子量＜3 kDa的降糖肽,并通过MTT法分析其对人肝癌细胞（HepG2细胞）的抑制作用。结果表明,酶法制备辣木籽降糖肽的最佳酶解时间为4.6 h、液料比40.5:1、pH为8.3、酶添加量5.5%、温度55 ℃,该条件下酶解产物的α-葡萄糖苷酶抑制率为23.62%±0.14%。超滤组分中分子量<3 kDa组分的α-葡萄糖苷酶抑制率为IC₅₀值为5.56 mg/mL,当其质量浓度为300 μg/mL时,作用于HepG2细胞48 h后能显著抑制HepG2细胞的增殖（P<0.05）。研究可为辣木籽降糖肽的进一步分离纯化奠定基础。     

壳聚糖固定化α-葡萄糖苷酶的研究

以粉末状壳聚糖为载体 ,采用吸附 交联的方法将α 葡萄糖苷酶固定化。在最适固定化条件下 ,室温吸附 6h ,然后与 3 5%的戊二醛在 4 5℃交联 6h ,可得到固定化酶的活力为1430 0U ,酶活力回收率为 59 6 %。通过实验发现 ,与游离酶相比 ,固定化酶的最适 pH向酸性方向移动 0 5pH单位 ,为 pH 4 5;最适作用温度达到 70℃ ,比游离α 葡萄糖苷酶提高 5℃ ;酸碱稳定性、热稳定性及贮存稳定性均有较大提高 ;在 6 0℃操作半衰期为 16 8h     

A Novel α-Glucosidase of the Glycoside Hydrolase Family 31 from Aspergillus sojae

We characterized an α-glucosidase belonging to the glycoside hydrolase family 31 from Aspergillus sojae. The α-glucosidase gene was cloned using the whole genome sequence of A. sojae, and the recombinant enzyme was expressed in Aspergillus nidulans. The enzyme was purified using affinity chromatography. The enzyme showed an optimum pH of 5.5 and was stable between pH 6.0 and 10.0. The optimum temperature was approximately 55 °C. The enzyme was stable up to 50 °C, but lost its activity at 70 °C. The enzyme acted on a broad range of maltooligosaccharides and isomaltooligosaccharides, soluble starch, and dextran, and released glucose from these substrates. When maltose was used as substrate, the enzyme catalyzed transglucosylation to produce oligosaccharides consisting of α-1,6-glucosidic linkages as the major products. The transglucosylation pattern with maltopentaose was also analyzed, indicating that the enzyme mainly produced oligosaccharides with molecular weights higher than that of maltopentaose and containing continuous α-1,6-glucosidic linkages. These results demonstrate that the enzyme is a novel α-glucosidase that acts on both maltooligosaccharides and isomaltooligosaccharides, and efficiently produces oligosaccharides containing continuous α-1,6-glucosidic linkages.     

α-Glucosidase Inhibitory Activity of Bromophenol Purified from the Red Alga Polyopes lancifolia

Abstract: A bromophenol, bis(2,3-dibromo-4,5-dihydroxybenzyl) ether, was purified from the red alga Polyopes lancifolia. Its IC₅₀ values were 0.098 and 0.120 μM against Saccharomyces cerevisiae and Bacillus stearothermophilus α-glucosidases, respectively, and 1.00 and 1.20 mM against rat-intestinal sucrase and maltase. This bromophenol competitively inhibited S. cerevisiae α-glucosidase with a K_I value of 0.068 μM and was very stable at pH 2 for 60 min at 37 °C. Therefore, this P. lancifolia bromophenol may have potential as natural nutraceutical for the management of type 2 diabetes. Practical Application: One therapeutic approach for preventing diabetes mellitus is to retard the absorption of glucose via inhibition of α-glucosidase. In this study, one compound with strong α-glucosidase-inhibitory activity, bis(2,3-dibromo-4,5-dihydroxybenzyl) ether, was purified and identified from the red alga P. lancifolia. Therefore, P. lancifolia bromophenol can potentially be developed as a novel natural nutraceutical for the management of type 2 diabetes.