Cassava pullulanase and its synergistic debranching action with isoamylase 3 in starch catabolism

Pullulanase (EC 3.2.1.41, PUL), a debranching enzyme belonging to glycoside hydrolase family 13 subfamily 13, catalyses the cleavage of α-1,6 linkages of pullulan and β-limit dextrin. The present work studied PUL from cassava Manihot esculenta Crantz (MePUL) tubers, an important economic crop. The Mepul gene was successfully cloned and expressed in E. coli and rMePUL was biochemically characterised. MePUL was present as monomer and homodimer, as judged by apparent mass of ~ 84 - 197 kDa by gel permeation chromatography analysis. Optimal pH and temperature were at pH 6.0 and 50 °C, and enzyme activity was enhanced by the addition of Ca2+ ions. Pullulan is the most favourable substrate for rMePUL, followed by β-limit dextrin. Additionally, maltooligosaccharides were potential allosteric modulators of rMePUL. Interestingly, short-chain maltooligosaccharides (DP 2 - 4) were significantly revealed at a higher level when rMePUL was mixed with cassava isoamylase 3 (rMeISA3), compared to that of each single enzyme reaction. This suggests that MePUL and MeISA3 debranch β-limit dextrin in a synergistic manner, which represents a major starch catabolising process in dicots. Additionally, subcellular localisation suggested the involvement of MePUL in starch catabolism, which normally takes place in plastids

Computational design of Lactobacillus Acidophilus α-L-rhamnosidase to increase its structural stability.

α-L-rhamnosidase catalyzes hydrolysis of the terminal α-L-rhamnose from various natural rhamnoglycosides, including naringin and hesperidin, and has various applications such as debittering of citrus juices in the food industry and flavonoid derhamnosylation in the pharmaceutical industry. However, its activity is lost at high temperatures, limiting its usage. To improve Lactobacillus acidophilus α-L-rhamnosidase stability, we employed molecular dynamics (MD) to identify a highly flexible region, as evaluated by its root mean square fluctuation (RMSF) value, and computational protein design (Rosetta) to increase rigidity and favorable interactions of residues in highly flexible regions. MD results show that five regions have the highest flexibilities and were selected for design by Rosetta. Twenty-one designed mutants with the best ΔΔG at each position and ΔΔG < 0 REU were simulated at high temperature. Eight designed mutants with ΔRMSF of highly flexible regions lower than -10.0% were further simulated at the optimum temperature of the wild type. N88Q, N202V, G207D, Q209M, N211T and Y213K mutants were predicted to be more stable and could maintain their native structures better than the wild type due to increased hydrogen bond interactions of designed residues and their neighboring residues. These designed mutants are promising enzymes with high potential for stability improvement

The Intake of Energy and Selected Nutrients by Thai Urban Sedentary Workers: An Evaluation of Adherence to Dietary Recommendations

Rapid changes in Thailand’s nutrition and lifestyles have led to increasing diet-related pathologies among people with sedentary occupations. This study examines the extent to which the dietary intake of nutrients and energy by a sample of Thai sedentary workers conforms to the Thai Dietary Reference Intakes (Thai DRIs). The nutrients and energy intake estimates were based on self-reported information collected with a single 24-hour dietary recall and nonweighed 2-day food record. The study participants were Thai adults aged 20–50 years employed in sedentary occupations. A convenience sample of 215 healthy individuals (75 males and 140 females) was based on four randomly selected worksites in the Bangkok metropolitan area. For male participants, the study found a median energy intake of 1,485 kcal/day, with 54.4% of energy coming from carbohydrate, 15.9% from protein, and 29.6% from fat. Females’ median energy intake was 1,428 kcal/day, 56% of which came from carbohydrate, 16.2% from protein, and 28.6% from fat. Both genders showed insufficient intake of fiber and most micronutrients. This study provides the material for preventive public health interventions focusing on nutrition-related diseases affecting Thailand’s rapidly growing sedentary workforce

The diagnostic tests and functional outcomes of acute ischemic stroke or transient ischemic attack in young adults: A 4-year hospital-based observational study.

Background and objectivesIschemic strokes in young adults have been a significant concern due to various potential etiologies and had substantial clinical and public health impacts. We aimed to study the diagnostic tests, etiologies, and functional outcomes of acute ischemic stroke (AIS) and transient ischemic attack (TIA) in young adult patients.MethodsThe data were retrieved from the Chiang Mai University Hospital Stroke Registry between January 2018 and December 2021. Consecutive AIS or TIA patients were included if they were 18-50 years and had no stroke mimics. Study outcomes were proportions of positive diagnostic tests, and 90-day modified Rankin Scale (mRS).ResultsOf 244 enrolled patients, 59.0% (n = 144) were male, and 38.1% (n = 93) were aged 18-40, classified as the younger age group. There was a high incidence of diabetes (24.5%) and dyslipidemia (54.3%) among patients aged 41-50, associated with small-vessel occlusion and large-artery atherosclerosis stroke classification in this age group. Patients aged 18-40 years had more other determined etiologies (39.8%), with hypercoagulability (8.2%), arterial dissection (7.8%), and cardiac sources (6.6%) being the first three causes, which were associated with higher anticoagulant treatment. The cerebrovascular study, cardiac evaluation using echocardiography, and antiphospholipid syndrome testing were commonly performed, of which computed tomography angiography provided a high proportion of positive results (80.3%). 76.3% of young adult patients had excellent functional outcomes (mRS 0-1) with a median mRS of 0 (interquartile range 0-1) at 90-day follow-up.ConclusionsStroke of other determined etiology remained the common cause of stroke in young adults, and most affected individuals had excellent clinical outcomes. Blood tests for arterial hypercoagulability and noninvasive vascular and cardiac evaluations are encouraged in selected patients to determine the stroke etiology and guide for appropriate preventive strategies

Conserved Calcium-Binding Residues at the Ca-I Site Involved in Fructooligosaccharide Synthesis by Lactobacillus reuteri 121 Inulosucrase

Inulosucrase is an enzyme that synthesizes inulin-type β-2,1-linked fructooligosaccharides (IFOS) from sucrose. Previous studies have shown that calcium is important for the activity and stability of Lactobacillus reuteri 121 inulosucrase (LrInu). Here, mutational analyses of four conserved calcium-binding site I (Ca-I) residues of LrInu, Asp418, Gln449, Asn488, and Asp520 were performed. Alanine substitution for these residues not only reduced the stability and activity of LrInu, but also modulated the pattern of the IFOS produced. Circular dichroism spectroscopy and molecular dynamics simulation indicated that these mutations had limited impact on the overall conformation of the enzyme. One of Ca-I residues most critical for controlling LrInu-mediated polymerization of IFOS, Asp418, was also subjected to mutagenesis, generating D418E, D418H, D418L, D418N, D418S, and D418W. The activity of these mutants demonstrated that the IFOS chain length could be controlled by a single mutation at the Ca-I site

Rational re-design of Lactobacillus reuteri 121 inulosucrase for product chain length control

Fructooligosaccharides (FOSs) are well-known prebiotics that are widely used in the food, beverage and pharmaceutical industries. Inulosucrase (E.C. 2.4.1.9) can potentially be used to synthesise FOSs from sucrose. In this study, inulosucrase from Lactobacillus reuteri 121 was engineered by site-directed mutagenesis to change the FOS chain length. Three variants (R483F, R483Y and R483W) were designed, and their binding free energies with 1,1,1-kestopentaose (GF4) were calculated with the Rosetta software. R483F and R483Y were predicted to bind with GF4 better than the wild type, suggesting that these engineered enzymes should be able to effectively extend GF4 by one residue and produce a greater quantity of GF5 than the wild type. MALDI-TOF MS analysis showed that R483F, R483Y and R483W variants could synthesise shorter chain FOSs with a degree of polymerization (DP) up to 11, 10, and 10, respectively, while wild type produced longer FOSs and in polymeric form. Although the decrease in catalytic activity and the increase of hydrolysis/transglycosylation activity ratio was observed, the variants could effectively synthesise FOSs with the yield up to 73% of substrate. Quantitative analysis demonstrated that these variants produced a larger quantity of GF5 than wild type, which was in good agreement with the predicted binding free energy results. Our findings demonstrate the success of using aromatic amino acid residues, at position D418, to block the oligosaccharide binding track of inulosucrase in controlling product chain length