Enzyme engineering of fungal-derived FAD-GDH by circular permutation

The flavin adenine dinucleotide dependent glucose dehydrogenase (FAD-GDH; EC 1.1.5.9) comprises oxidoreductases that catalyze the initial oxidation of glucose and other sugar molecules, using FAD as the primary electron acceptor. FAD-GDH has received attention as biocatalyst for glucose monitoring, especially self-monitoring of blood glucose. Please click Additional Files below to see the full abstract

Protein engineering of Candida rugosa lipase

Lipases (EC 3.1.1.3) catalyze the hydrolysis of emulsified long-chain triacylglycerol at the lipid–water interface. Candida rugosa lipase (CRL) is one of important industrial enzymes that are widely used in biotechnological applications such as the production of fatty acids and the synthesis of various esters. The catalytic efficiency and substrate specificity are seemed to be the key factors for industrial applications. Several approaches have been used to increase the stability of different lipases. For example, the immobilized CRL on carriers led to significant improvement in the catalyst’s activity and stability (Ref 1). In this study, we have focused on protein engineering to improve the properties of CRL. Please click Additional Files below to see the full abstract

Enhancement of protein thermostability by three consecutive mutations using loop-walking method and machine learning

We developed a method to improve protein thermostability, "loop-walking method". Three consecutive positions in 12 loops of Burkholderia cepacia lipase were subjected to random mutagenesis to make 12 libraries. Screening allowed us to identify L7 as a hot-spot loop having an impact on thermostability, and the P233G/L234E/V235M mutant was found from 214 variants in the L7 library. Although a more excellent mutant might be discovered by screening all the 8000 P233X/L234X/V235X mutants, it was difficult to assay all of them. We therefore employed machine learning. Using thermostability data of the 214 mutants, a computational discrimination model was constructed to predict thermostability potentials. Among 7786 combinations ranked in silico, 20 promising candidates were selected and assayed. The P233D/L234P/V235S mutant retained 66% activity after heat treatment at 60 degrees C for 30 min, which was higher than those of the wild-type enzyme (5%) and the P233G/L234E/V235M mutant (35%)

A Novel Strategy in Production of Oligosaccharides in Digestive Tract: Prevention of Postprandial Hyperglycemia and Hyperinsulinemia

The aim of this study was to evaluate the effects of oral administration of transglucosidase (TG) on postprandial glucose concentrations in healthy subjects. A randomized placebo-controlled three-way crossover trial was separated by a washout period of more than 3 days. Twenty-one normal healthy volunteers, aged 30–61 years old (17 males and 4 females) were selected for this study. The subjects’ health was assessed as normal by prestudy screening. All subjects received 3 types of test meals (3 rice balls: protein, 14.4 g; fat, 2.1 g; and carbohydrate, 111 g: total energy, 522 kcal) with 200 ml water in which 0 mg, 150 mg, or 300 mg of TG was dissolved. Blood samples for estimating plasma glucose and insulin concentrations were collected before and every 30 min after the experiment. As compared to no TG treatment, TG administration tended to prevent a postprandial increase in plasma glucose (p = 0.069: 150 mg of TG vs control) but there were no significant difference among three groups. With regard to the 17 subjects who were suggested to have impaired glucose tolerance, TG significantly decreased the postprandial blood glucose (p<0.05: 150 mg and 300 mg of TG vs control) and marginally decreased insulin concentrations (p = 0.099: 300 mg of TG vs control). These results suggest that TG may be useful for preventing the progression of type 2 diabetes mellitus

Development of P450-BM3 using molecular dynamics simulations- A tribute to the late Professor Hideaki Yamada

Please click Additional Files below to see the full abstract

Supplementation With Whey Peptide Rich in β-Lactolin Improves Cognitive Performance in Healthy Older Adults: A Randomized, Double-Blind, Placebo-Controlled Study

Epidemiological reports showed that consumptions of fermented dairy products are beneficial for cognitive decline in elderly. Our previous preclinical studies have demonstrated that intakes of whey peptide rich in the β-lactolin [β-lactopeptide of glycine-thereonine-tryptophan-tyrosine (GTWY)] improve memory and attention by regulating monoamine system, and clinical study using neuropsychological test suggested that consumptions with GTWY-rich whey peptide enhance cognitive performance associated with the frontal cortex activity. However, corresponding interventional studies in humans are limited. Objectives: to evaluate the effects of the whey peptide on cognitive functions in healthy older adults using a randomized, double-blinded, placebo-controlled trial design. 114 healthy subjects aged 50–75 were supplemented with the whey peptide or placebo for 12 weeks, and changes in cognitive function were assessed using neuropsychological tests at weeks 0, 6, and 12 of the intervention. Neuropsychological tests included assessments for memory functions (subtests from Wechsler memory scale-revised, standard verbal paired-associate learning test, and recognition memory test for faces), assessments for attention (cancelation and detection tests), and assessments for general cognitive functions (repeatable battery for assessments of neuropsychological status). Cerebral blood flow was also assessed using near-infrared spectroscopy (NIRS) after 6 weeks of intervention. This study was registered on the 19 November, 2017 in the database of the University Hospital Medical Information Network (UMIN) prior to enrollment of subjects (Registration No. UMIN000030461: https://www.umin.ac.jp/ctr/index-j.htm). In the whey peptide group, visual paired-associates I and visual cancelation tests were significantly improved compared with those in the placebo group at weeks 6 and 12 of the intervention, respectively. Visuospatial and constructional scores of the repeatable battery for assessments of neuropsychological status and standard verbal paired-associate learning tests (S-PA) also tended to be improved by the intervention at week 12. Daily intakes of GTWY-rich whey peptide show beneficial effects on cognitive performance, especially associative learning memory and control of attention, in healthy older adults and might prevent age-related cognitive declines

Monobody-Mediated Alteration of Lipase Substrate Specificity

Controlling the catalytic properties of enzymes remain an important challenge in chemistry and biotechnology. We have recently established a strategy for altering enzyme specificity in which the addition of proxy monobodies, synthetic binding proteins, modulates the specificity of an otherwise unmodified enzyme. Here, in order to examine its broader applicability, we employed the strategy on <i>Candida rugosa</i> lipase 1 (CRL1), an enzyme with a tunnel-like substrate binding site. We successfully identified proxy monobodies that restricted the substrate specificity of CRL1 toward short-chain fatty acids. The successes with this enzyme system and a β-galactosidase used in the previous work suggest that our strategy can be applied to diverse enzymes with distinct architectures of substrate binding sites