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%)

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

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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

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

Characterisation of a Novel White Laccase from the Deuteromycete Fungus Myrothecium verrucaria NF-05 and Its Decolourisation of Dyes

A novel ‘white’ laccase was purified from the deuteromycete fungus, Myrothecium verrucaria NF-05, which was a high laccase-producing strain (40.2 U·ml−1 on the thirteenth day during fermentation). SDS-PAGE and native-PAGE revealed a single band with laccase activity corresponding to a molecular weight of approximately 66 kDa. The enzyme had three copper and one iron atoms per protein molecule determined by ICP-AES. Furthermore, both UV/visible and EPR spectroscopy remained silence, indicating the enzyme a novel laccase with new metal compositions of active centre and spectral properties. The N-terminal amino acid sequence of the purified protein was APQISPQYPM. Together with MALDI-TOF analysis, the protein revealed a high homology of the protein with that from reported M. verrucaria. The highest activity was detected at pH 4.0 and at 30°C. The enzyme activity was significantly enhanced by Na+, Mn2+, Cu2+ and Zn2+ while inhibited by DTT, NaN3 and halogen anions. The kinetic constant (Km) showed the enzyme was more affinitive to ABTS than other tested aromatic substrates. Twelve structurally different dyes could be effectively decolourised by the laccase within 10 min. The high production of the strain and novel properties of the laccase suggested its potential for biotechnological applications