Purification and characterization of actinidin from Actinidia deliciosa and its utilization in inactivation of α-amylase

Background Actinidin is an anionic thiol-proteinase predominant and unique to Chinese gooseberry or kiwifruit, whose strong digestibility enables proteins or enzymes vulnerable to digestion. The arrangement of active cysteine–thiol residues (Cys22-Cys65, Cys56-Cys98, and Cys156-Cys206) stabilizes the catalytic unit, thus allowing an effective Inhibition of α-amylase protein on exposure to the highest concentrations of actinidin under optimum conditions. When starch-rich foods are consumed with kiwifruit, starch digestion may be slowed by the inactivation of α-amylase (digestive enzyme), specifically reducing the blood sugar levels by hindering starch digestion that is helpful in diabetes mellitus. Thus, the study aimed at actinidin purification, optimization for maximal activity, and its demonstration as a potential to degrade α-amylase. Results Protease showed a molecular mass of 27 kDa on SDS-PAGE analysis. One factor at a time method was applied for process optimization, increasing the actinidin yield up to 176.03 U/mg. The enzyme was stable at a wide pH range; however, it was most active and stable at pH 7.5. The enzyme possessed half‐life at 35 °C of 5.5 h, at 40 °C of 4.5 h, at 45 °C of 2.5 h, and at 50 °C of 1 h. Lineweaver–Burk plot showed Michaelis–Menten constant (Km: 3.14 mg/ml) and maximal velocity (Vmax: 1.428 mmol/ml/min) using casein. The actinidin activity was enhanced with Ca2+ while it was inhibited by Cd2+ and Hg2+ ions. The α-amylase protein was successfully inactivated upon incubation with actinidin for 30 min; around ~ 85% of the α-amylase activity diminished. IC50 for inhibition of α-amylase was 2.54 mg/ml for crude actinidin and 1.86 mg/ml for purified actinidin. Conclusions Purified Actinidin showed a 1.28-fold increase in proteolytic activity. The proteinase showed an active pH range of 3.5–8.5 under varied buffer conditions and thermostability up to 50 °C. The results revealed a significant potential utility of actinidin to retard amylase as it effectively degraded the amylolytic enzyme under in vitro conditions and could be beneficial for lowering glycemic response to ingested starch. However, further in vitro as well as in vivo studies need to be conducted under gastrointestinal conditions to establish the hypothesis.

Influence of Extracts from Bark of Deciduous Trees on the Activity of the Amylolytic Enzyme - Alpha Amylase

The effect of water extracts obtained from bark of growing in Latvia and widely spread in Europe grey alder (Alnusincana), black alder (Alnusglutinosa) and aspen tree (Populustremula), on the amylase activity in human saliva was evaluated. The extracts were obtained using advanced ACE equipment and distilled hot water as a solvent. The total yields of extractives from bark were rather similar and varied from 16 % to 21 % based on dry bark. However, the content of proantocianidine and salicine derivatives used as diagnostic characteristic for evaluation of effect of extracts on activity of amylase – a glucose-polymers cleavage enzyme - differed significantly. The results of in-vitro tests realized using the model of human gastrointestinal pass have shown that the abovementioned extracts in micro dosages are able to increase significantly activity of amylase. However, this effect is dose dependent and when the dose is exceeded the opposite effect – inhibition of amylase activity - is observed. This effect is explained by increasing of proantocianidins component in the volume of the tested extract dose, because pure proantocianidins, isolated from grey alder bark significantly inhibited activity of amylase. The further investigation is necessary in order to estimate effective and safe dosages for application of extracts providing a guaranteed unambiguous effect of activation or inhibition of amylase activity.

Rapid sanitary and technological control of culturing the producers of amylolytic enzymes.

It has been established that the biotechnological process of culturing bacillary microbial producers of amylolytic enzymes can be express-controlled by determining their ATP bioluminescence. The advantages of the method have been shown. The analytical review of producers of hydrolytic enzymes has made it clear how practical it is to use bacillary microorganisms for targeted secretion of amylolytic enzymes in biotechnological production. After monitoring bacillary microorganisms, it has been found advisable to choose Bacillus subtilis ATCC 6633 as the working culture due to its time of production of amylolytic enzymes and its biosynthetic activity. Reasons have been given for using the rapid ATP control method, which is based on the principle of bioluminescence. Different growth media have been compared and evaluated in order to intensify the quantitative biosynthetic activity of the microbial culture in the technological process of culturing bacillary microorganisms. The experiments have proved that growth media can be modified by introducing a number of carbohydrate–protein substrates as inducers of amylolytic complex gene expression. The latter manifests itself in the amylolytic activity accelerated by 12–24 hours, and causes an increase in the number of microorganisms (1.87–3.99 times as many as in the reference culture). Two methods of control (rapid bioluminescent and classic microbiological) have been used for correlative determination of the quantitative growth of Bacillus subtilis cells. Mathematical straight-line correlations have been obtained in a semilogarithmic system for the number of cells of the bacillary producer of the amylolytic enzyme complex. These correlations allow carrying out rapid control in a production environment. Along with the traditional rapid sanitary control in biotechnological production, which includes controlling the contamination of the equipment, personnel’s hands, and water, it has been suggested to perform proprietary technological express control of amylolytic enzyme biosynthesis using the culture Bacillus subtilis ATCC 6633

SKRINING BAKTERI TERMOFILIK POTENSIAL AMILOLITIK DARI SUMBER AIR PANAS WAY BELERANG KALIANDA LAMPUNG SELATAN

Thermophilic bacteria that produced amylase and protease have been isolated from Way Belerang hot spring, Kalianda, South Lampung. This research aims to screen and identify thermophilic bacteria that have the potential to produce thermostable amylase and protease enzymes.The research procedures included sampling, isolation of enzyme-producing thermophilic bacteria, a series of phenotypic and biochemical tests, and molecular identification by 16s rRNA. This study used 2 treatments, namely incubation temperature 37 and 50 ºC with 3 repetitions. The results showed that the optimum temperature for growth of thermophilic bacterial isolates and thermophilic bacterial isolates producing amylase enzymes was 50ºC. The bacteria isolate that had the best amylolytic enzyme activity was Isolate A.WB.50.1 with a diameter of the inhibitory zone was 15.44 mm. Isolate A.WB.50.1 has been identified by the species Pseudomonas stutzeri.

Effect of Different Steeping and Malting Regimen on the Amylolytic Activities of Some Improved Nigerian Sorghum Grain Varieties

Three Nigerian improved sorghum varieties were evaluated to ascertain how different steeping and malting regimen affect their amylolytic enzyme development. Steeping incorporated air rest and continuous steep regime for 72 h. Samples were withdrawn every 12 h. Germination was then carried out for four days before kilning at 50°C for 24 h. Grain and malt parameters were examined. Results obtained showed variations in the response of sorghum root length to steep regimen and time. CSR-02 gave maximum root length (3.32 cm) after 72 h of air rested steeping. CSR-02, Samsorg 44 and Samsorg 14 had germinative energies of 92.00 ± 4.24, 94.00 ± 1.41 and 96.00 ± 1.41%; germinative capacities of 91.00 ± 1.41, 75.50 ± 2.12 and 88.00 ± 2.83; water sensitivities of 6.50 ± 2.12, 13.50 ± 1.44 and 1.00 ± 0.41 respectively. TKW results were 29.73 ± 0.32, 33.85 ± 1.54 and 33.51 ± 0.41 kg for CSR-02, Samsorg 44 and Samsorg 14 respectively. Variations in the response of the sorghum varieties to various conditions of steep regime and steep period were also observed. Steeping for 48 h seems to be the optimum time for the development of amylolytic activity in all the sorghum varieties at both steeping regimens. Samsorg 14 gave the highest total amylase activity (355.44 µg glucose equivalents), followed by Samsorg 44 (278.08 µg glucose equivalents). Samsorg 14 also showed the highest α-amylase development (276.93 µg glucose equivalents). Air rest was found to be show greater effect on β-amylase development in all the sorghum varieties.

Influence of heat treatment in the presence of certain metal ions on the spectral-fluorescent properties of an amylolytic enzyme preparation

Тепловая обработка ферментных препаратов приводит к заметному изменению их активности в результате определенных конформационных изменений в молекуле фермента. На основе изучения ультрафиолетовых спектров авторы судят об изменении полярности окружения остатков триптофана при добавлении ионов цинка, алюминия и кальция. В работе приведены результаты изучения спектров флуоресценции ферментного препарата – ультраконцентрата глюкоамилазы Asp. awamori после тепловой обработки и добавления ионов некоторых металлов для обнаружения структурных изменений в молекуле фермента при этих воздействиях. Установлено, что совместное влияние ионов металлов и теплового воздействия на водный раствор ферментного препарата ультраконцентрата глюкоамилазы Asp. awamori приводит к повышению интенсивности свечения и смещению λmax в спектрах флуоресценции ультраконцентрата глюкоамилазы Asp. awamori в область более коротких длин волн. Это указывает на увеличение гидрофобности окружения триптофана и подтверждает конформационные изменения в молекуле фермента, происходящие под действием ионов некоторых металлов и при тепловом воздействии. The heat treatment of enzyme preparations leads to a noticeable change in their activity as a result of certain conformational changes in the enzyme molecule. On mastering the study of ultraviolet spectra, the authors judge a change in the polarity of the environment of tryptophan residues with the addition of zinc, aluminum and calcium ions. The paper presents the results of studying the fluorescence spectra of an enzyme preparation: glucoamylase ultraconcentrate Asp. awamori after heat treatment and the addition of certain ions of some metals to detect structural changes in the enzyme molecule under these influences. It was found that the combined effect of metal ions and thermal effects on the aqueous solution of the enzyme preparation of glucoamylase ultraconcentrate Asp. awamori, leads to an increase in luminescence intensity and a shift of λmax in the fluorescence spectra of Asp. awamori glucoamylase ultraconcentrate to the region of shorter wavelengths. This indicates an increase in the hydrophobicity of the tryptophan environment and provides confirmation of conformational changes in the enzyme molecule under the action of ions of certain metals and upon thermal exposure.