Monobody-mediated alteration of enzyme specificity.

Current methods for engineering enzymes modify enzymes themselves and require a detailed mechanistic understanding or a high-throughput assay. Here, we describe a new approach where catalytic properties are modulated with synthetic binding proteins, termed monobodies, directed to an unmodified enzyme. Using the example of a ß-galactosidase from Bacillus circulans, we efficiently identified monobodies that restricted its substrates for its transgalactosylation reaction and selectively enhanced the production of small oligosaccharide prebiotics.

Synthetically useful variants of industrial lipases from Burkholderia cepacia and Pseudomonas fluorescens.

Industrial enzymes lipase PS (LPS) and lipase AK (LAK), which originate from Burkholderia cepacia and Pseudomonas fluorescens, respectively, are synthetically useful biocatalysts. To strengthen their catalytic performances, we introduced two mutations into hot spots of the active sites (residues 287 and 290). The LPS_L287F/I290A double mutant showed high catalytic activity and enantioselectivity for poor substrates for which the wild-type enzyme showed very low activity. The LAK_V287F/I290A double mutant was also an excellent biocatalyst with expanded substrate scope, which was comparable to the LPS_L287F/I290A double mutant. Thermodynamic parameters were determined to address the origin of the high enantioselectivity of the double mutant. The ΔΔH‡ term, but not the ΔΔS‡ term, was predominant, which suggests that the enantioselectivity is driven by a differential energy associated with intermolecular interactions around Phe287 and Ala290. A remarkable solvent effect was observed, giving a bell-shaped profile between the E values and the log P or ε values of solvents with the highest E value in i-Pr2O. This suggests that an organic solvent with appropriate hydrophobicity and polarity provides the double mutant with some flexibility that is essential for excellent catalytic performance.

Transglucosidase improves the gut microbiota profile of type 2 diabetes mellitus patients: a randomized double-blind, placebo-controlled study.

BACKGROUND: Recently, the relationship between gut microbiota and obesity has been highlighted. The present randomized, double-blind, placebo-controlled study aimed to evaluate the efficacy of transglucosidase (TGD) in modulating blood glucose levels and body weight gain in patients with type 2 diabetes mellitus (T2DM) and to clarify the underlying mechanism by analyzing the gut microbiota of T2DM patients. METHODS: This study included 60 patients who received placebo or TGD orally (300 or 900 mg/day) for 12 weeks, and blood and fecal samples were collected before and after 12 weeks. Comparisons of fecal bacterial communities were performed before and after the TGD treatment and were performed between T2DM patients and 10 healthy individuals, using the terminal-restriction fragment length polymorphism analysis. RESULTS: The Clostridium cluster IV and subcluster XIVa components were significantly decreased, whereas the Lactobacillales and Bifidobacterium populations significantly increased in the T2DM patients compared with the healthy individuals. By dendrogram analysis, most of the healthy individuals (6/10) and T2DM patients (45/60) were classified into cluster I, indicating no significant difference in fecal bacterial communities between the healthy individuals and the T2DM patients. In the placebo and TGD groups, the bacterial communities were generally similar before and after the treatment. However, after 12 weeks of TGD therapy, the Bacteroidetes-to-Firmicutes ratio in the TGD groups significantly increased and was significantly higher compared with that in the placebo group, indicating that TGD improved the growth of the fecal bacterial communities in the T2DM patients. CONCLUSIONS: Therefore, TGD treatment decreased blood glucose levels and prevented body weight gain in the T2DM patients by inducing the production of oligosaccharides in the alimentary tract and modulating gut microbiota composition. TRIAL REGISTRATION: UMIN-CTR UMIN000010318.

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 °C for 30 min, which was higher than those of the wild-type enzyme (5%) and the P233G/L234E/V235M mutant (35%).

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 Candida rugosa 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 ß-galactosidase used in the previous work suggest that our strategy can be applied to diverse enzymes with distinct architectures of substrate binding sites.

Combination of Gluten-Digesting Enzymes Improved Symptoms of Non-Celiac Gluten Sensitivity: A Randomized Single-blind, Placebo-controlled Crossover Study.

INTRODUCTION: Recently, the population of individuals with non-celiac gluten sensitivity (NCGS) who do not have celiac disease but show improved symptoms with a gluten-free diet, has increased. Enzyme replacement therapy using digestive enzymes is expected to improve the symptoms of NCGS and be sustainable, since gluten-related proteins that are indigestible by the digestive system have been considered triggers of NCGS. METHODS: We selected patients with NCGS by screening demographic interviews, as well as performing medical evaluations, anti-gluten antibody tests, and gluten challenge tests. We performed a single-blind and crossover clinical trial with these subjects using a gluten challenge with the enzyme mixture or a placebo. Our designed enzyme mixture contained peptidase, semi alkaline protease, deuterolysin, and cysteine protease derived from Aspergillus oryzae, Aspergillus melleus, Penicillium citrinum, and Carica papaya L., respectively. RESULTS: Administration of the enzyme mixture significantly decreased the change in the score of the symptom questionnaire before and after the gluten challenge compared with administration of the placebo in patients with NCGS without adverse events. In particular, the changes in the score of the gluten-induced incomplete evacuation feeling and headaches were significantly improved. The serum levels of interleukin (IL)-8, tumor necrosis factor (TNF)-α, andregulated on activation, normal T cell expressed and secreted (RANTES) in subjects were not significantly changed by gluten, as expected from previous studies, and the enzyme mixture did not affect these inflammatory markers. CONCLUSION: In this human clinical study, we demonstrated the efficacy of the enzyme mixture derived from microorganisms and papaya in improving the symptoms of NCGS.

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.

Transglucosidase improves the bowel movements in type 2 diabetes mellitus patients: A preliminary randomized double-blind, placebo-controlled study.

BACKGROUND: Recent studies have highlighted the relationship between gut microbiota and bowel movements. OBJECTIVE: We aimed to evaluate transglucosidase treatment efficacy for bowel movements in patients with type 2 diabetes mellitus and to clarify the relationship between bowel movements, dietary habits, gut microbiota and fecal short-chain fatty acids. METHODS: In this randomized double-blind, placebo-controlled study, 66 patients received placebo or transglucosidase (300 or 900 mg/day) orally, for 12 weeks. Fecal bacterial communities and short-chain fatty acids were analyzed before and after the treatment. RESULTS: Transglucosidase treatment significantly (p < 0.05) affected fecal microbiota (Prevotella spp., Bacteroides spp., Bifidobacterium spp., and Clostridium subcluster XIVa) and fecal short-chain fatty acid (acetate, valerate, succinate and lactate) content. Clostridium cluster IV, Clostridium subcluster XIVa, Clostridium cluster XVIII and fecal pH increased significantly and order Lactobacillales decreased in patients with bowel movement disorder compared with controls. Transglucosidase treatment significantly improved bowel movements compared with placebo treatment (46.2%, 95% confidence interval: 19.2-74.9% vs. 0%, 95% confidence interval: 0-33.6%, p < 0.05). This effect was not observed in patients without bowel movement disorder. CONCLUSION: Patients with bowel movement disorder suffer from gut dysbiosis. Transglucosidase treatment alleviates bowel movement disorder symptoms in type 2 diabetes mellitus patients by increasing fecal acetate level.

Improvement of Bacillus circulans beta-amylase activity attained using the ancestral mutation method.

Thermostabilization of enzymes is one of the greatest challenges of protein engineering. The ancestral mutation method, which introduces ancestral residues into a target enzyme, has previously been developed and used to improve the thermostabilities of thermophilic enzymes. Herein, we report a study that used the ancestral mutation method to improve the thermostability of Bacillus circulans beta-amylase, a mesophilic enzyme. A bacterial, common-ancestral beta-amylase sequence was inferred using a phylogenetic tree composed of higher plant and bacterial amylase sequences. Eighteen mutants containing ancestral residues were designed, expressed in Escherichia coli and purified. Several of these mutants were more thermostable than that of the wild-type amylase. Notably, one mutant had both greater activity and greater thermostability. The relationship between the extent to which the amino acid residues within 5 A of the mutation site were evolutionarily conserved and the extent to which thermostability was improved was examined. Apparently, it is necessary to conserve the residues surrounding an ancestral residue if thermostability is to be improved by the ancestral mutation method.

Supplementing transglucosidase with a high-fiber diet for prevention of postprandial hyperglycemia in streptozotocin-induced diabetic dogs.

Indigestible oligosaccharides have been shown to normalize blood glucose and insulin concentration thereby promoting good health and preventing diseases, such as diabetes. Transglucosidase (TG, alpha-glucosidase, enzyme code (EC) 3.2.1.20) is an enzyme capable of converting starch to oligosaccharides, such as iso-malto-oligosaccharides from maltose, via the action of amylase. The aim of this study was to evaluate whether oral administration of TG with maltose or dextrin is capable of reducing post-prandial serum glucose concentration in experimentally streptozotocin (STZ)-induced diabetic dogs fed on a high-fiber diet. Five healthy and five STZ-induced diabetic dogs were employed in this study. TG supplementation with dextrin or maltose had no detrimental effect in healthy dogs. In fact, TG and dextrin exhibited a flatlined serum glucose pattern, while reducing mean post-prandial serum insulin and glucose concentration as compared to control diet alone. When TG supplementation was tested in STZ-induced diabetic dogs under the context of a high fiber diet, a 13.8% and 23.9% reduction in mean glucose concentration for TG with maltose and dextrin, respectively was observed. Moreover, TG with dextrin resulted in a 13% lower mean post-prandial glucose concentration than TG with maltose, suggesting that dextrin may be a more efficient substrate than maltose when used at the same concentration (1 g/kg). Our results indicate that TG supplementation with diet can lead to lower postprandial glucose levels versus diet alone. However, the efficacy of TG supplementation may depend on the type of diet it is supplemented with. As such, TG administration may be useful for preventing the progression of diabetes mellitus and in its management in dogs.