ABSTRACT
The development of an efficient screening method for the activity of PET-degrading enzymes represents a significant technological advance in the field of enzyme research, with the potential to facilitate the advancement of enzymes for PET recycling. By examining the stable conditions of PET suspension and enzyme production conditions, we developed a method to quantify PET-degrading enzyme activity in E. coli culture medium using turbidity reduction as an indicator. High PET concentration or ionic strength caused aggregation of PET, and the best condition for activity detection was 0.5 mg mL-1 PET in 50 mM sodium phosphate pH 7.0. Preculture of E. coli increased the purity of enzyme secreted in medium. To evaluate the screening method, 720 colonies of the PET2-7M-H229X-F233X mutant library were analyzed and three candidates of high-activity mutants were obtained. The thermostability of the mutants could also be easily measured by measuring the residual activity after heat treatment. The H229T-F233M mutant showed 3.4 times higher degradation rate against PET film than the template enzyme at the initial time. The molecular dynamics simulation implied that the F233M mutation makes space for making an α helix and that the H229T mutation resolved the steric hindrance with Trp199. These mutations were speculated to change the angle of the Trp199 side chain of PET2 to an angle similar to that of the Trp185 of IsPETase, making it suitable for PET binding to the active center. Screening of activity using PET suspensions is compatible with robotic automation and is expected to be useful for validating computationally predicted mutations.
ABSTRACT
The bulky phenolic compound tetrabromobisphenol A (TBBPA) is a brominated flame retardant used in a wide range of products; however, it diffuses into the environment, and has been reported to have toxic effects. Although it is well-known that white-rot fungi degrade TBBPA through ligninolytic enzymes, no other metabolic enzymes have yet been identified, and the toxicity of the reaction products and their risks have not yet been examined. We found that the white-rot fungus Phanerochaete sordida YK-624 converted TBBPA to TBBPA-O-ß-D-glucopyranoside when grown under non-ligninolytic-enzyme-producing conditions. The metabolite showed less cytotoxicity and mitochondrial toxicity than TBBPA in neuroblastoma cells. From molecular biological and genetic engineering experiments, two P. sordida glycosyltransferases (PsGT1c and PsGT1e) that catalyze the glycosylation of TBBPA were newly identified; these enzymes showed dramatically different glycosylation activities for TBBPA and bisphenol A. The results of computational analyses indicated that the difference in substrate specificity is likely due to differences in the structure of the substrate-binding pocket. It appears that P. sordida YK-624 takes up TBBPA, and reduces its cytotoxicity via these glycosyltransferases.
Subject(s)
Phanerochaete , Polybrominated Biphenyls , Biotransformation , Phanerochaete/metabolism , Polybrominated Biphenyls/metabolism , Glycosyltransferases/metabolismABSTRACT
The development of generic pharmaceuticals involves a bioequivalence study to ensure the therapeutic equivalence of the test formulation to the original innovative product. The formulation characteristics of generic products are expected to be maintained in the long term after approval. This study analyzed the factors contributing to the changes in the dissolution profiles of approved products during their life cycles. Cumulative data on the dissolution similarity of 1675 products of 127 ingredients tested by official laboratories in Japan were assessed according to Japanese bioequivalence guidelines with slight modifications. The products showing dissimilarities in dissolution profiles were analyzed for reporting year, therapeutic category, co-development, physical properties of the active pharmaceutical ingredient (API), and suspected reasons for dissolution change. The increase in the number of dissimilar products is related to the co-development of generic products. Although the solubility of the API was not associated with the dissolution change in the analysis of the total dissolution data, control of the API particle size is suggested to be important for drugs with poorly soluble APIs. Additionally, a risk factor for dissolution changes in the test solutions at a certain pH was the presence of acidic or basic residues. These results indicate the importance of proper development through a thorough evaluation of the formulation and process factors affecting the dissolution properties throughout the product lifecycle.
Subject(s)
Drugs, Generic , Therapeutic Equivalency , Solubility , Drugs, Generic/chemistry , JapanABSTRACT
Processive movement is the key reaction for crystalline polymer degradation by enzyme. Product release is an important phenomenon in resetting the moving cycle, but how it affects chitinase kinetics was unknown. Therefore, we investigated the effect of diacetyl chitobiose (C2) on the biochemical activity and movement of chitinase A from Serratia marcescens (SmChiA). The apparent inhibition constant of C2 on crystalline chitin degradation of SmChiA was 159 µM. The binding position of C2 obtained by X-ray crystallography was at subsite +1, +2 and Trp275 interact with C2 at subsite +1. This binding state is consistent with the competitive inhibition obtained by biochemical analysis. The apparent inhibition constant of C2 on the moving velocity of high-speed (HS) AFM observations was 330 µM, which is close to the biochemical results, indicating that the main factor in crystalline chitin degradation is also the decrease in degradation activity due to inhibition of processive movement. The Trp275 is a key residue for making a sliding intermediate complex. SmChiA W275A showed weaker activity and affinity than WT against crystalline chitin because it is less processive than WT. In addition, biochemical apparent inhibition constant for C2 of SmChiA W275A was 45.6 µM. W275A mutant showed stronger C2 inhibition than WT even though the C2 binding affinity is weaker than WT. This result indicated that Trp275 is important for the interaction at subsite +1, but also important for making sliding intermediate complex and physically block the rebinding of C2 on the catalytic site for crystalline chitin degradation.
Subject(s)
Chitinases , Chitinases/chemistry , Chitinases/metabolism , Chitin/chemistry , Chitin/metabolism , Catalytic Domain , Protein Binding , Serratia marcescens/metabolismABSTRACT
Preservatives are frequently added to cosmetics to maintain product quality. Our laboratory quantifies 11 preservatives in cosmetics each year for regulatory purposes. In laboratories, many manufactures also analyze the preservatives in their products for quality control. To analyze many cosmetic samples, a rapid analysis method is required for efficiency. In this study, we developed a rapid method for the simultaneous determination of 11 regulated preservatives in cosmetics using a core-shell column by high-performance liquid chromatography. In this method, the 11 preservatives were separated within 17 min, which was approximately half the time reported in the previous study. The peak resolution for each preservative was >2.6, the correlation coefficients of the calibration curves were >0.9988, the percent recoveries were 92.0-111.9% and the relative standard deviations were <3.5% (n = 3). The relative standard deviations among 6 researchers were <4.7%. Thus, it is an effective rapid determination method for the analysis of preservatives in cosmetics.
Subject(s)
Cosmetics , Preservatives, Pharmaceutical , Preservatives, Pharmaceutical/analysis , Cosmetics/analysis , Cosmetics/chemistry , Chromatography, High Pressure Liquid/methods , CalibrationABSTRACT
Efficient depolymerization of crystalline cellulose requires cooperation between multiple cellulolytic enzymes. Through biochemical approaches, molecular dynamics (MD) simulation, and single-molecule observations using high-speed atomic force microscopy (HS-AFM), we quantify and track synergistic activity for cellobiohydrolases (CBHs) with a lytic polysaccharide monooxygenase (LPMO) from Phanerochaete chrysosporium. Increasing concentrations of LPMO (AA9D) increased the activity of a glycoside hydrolase family 6 CBH, Cel6A, whereas the activity of a family 7 CBH (Cel7D) was enhanced only at lower concentrations of AA9D. MD simulation suggests that the result of AA9D action to produce chain breaks in crystalline cellulose can oxidatively disturb the crystalline surface by disrupting hydrogen bonds. HS-AFM observations showed that AA9D increased the number of Cel7D molecules moving on the substrate surface and increased the processivity of Cel7D, thereby increasing the depolymerization performance, suggesting that AA9D not only generates chain ends but also amorphizes the crystalline surface, thereby increasing the activity of CBHs.
ABSTRACT
To determine the usefulness of lymphocyte-to-monocyte ratio (LMR), neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), and inflammatory response biomarker (IRB) score for predicting disease-specific survival and delayed cervical lymph node metastasis in early-stage oral squamous cell carcinoma (OSCC). We retrospectively analyzed 72 patients with early-stage OSCC. Receiver operating characteristic curve analysis was used to determine the cutoff values for LMR, NLR, and PLR. IRB score was determined as follows: high LMR, high NLR, and low PLR, which were each rated as 1. These scores were added to obtain IRB score (range: 0-3). From univariate analysis, gender, poor mode of invasion, and high IRB score were identified as significant risk factors for disease-specific survival. However, there were no independent factors for poor prognosis in multivariate analysis. On the other hand, for delayed cervical lymph node metastasis, poor mode of invasion, low LMR, high NLR, high PLR, and high IRB score were identified as significant risk factors from univariate analysis, and in multivariate analysis, poor mode of invasion and high IRB score were confirmed as independent risk factors. IRB score and mode of invasion are potentially independent risk factors for delayed cervical lymph node metastasis in early-stage OSCC.
Subject(s)
Carcinoma, Squamous Cell , Head and Neck Neoplasms , Mouth Neoplasms , Humans , Prognosis , Carcinoma, Squamous Cell/surgery , Carcinoma, Squamous Cell/pathology , Mouth Neoplasms/surgery , Lymphatic Metastasis , Squamous Cell Carcinoma of Head and Neck/surgery , Retrospective Studies , Lymphocytes/pathology , Neutrophils/pathology , Systemic Inflammatory Response SyndromeABSTRACT
INTRODUCTION: Clinical data of esaxerenone in hypertensive patients with diabetic kidney disease (DKD) are lacking. We evaluated the efficacy and safety of esaxerenone in patients with DKD and an inadequate response to blood pressure (BP)-lowering treatment. METHODS: In this multicenter, open-label, prospective study, patients were divided into urinary albumin-to-creatinine ratio subcohorts (UACR < 30, 30 to < 300, and 300 to < 1000 mg/gCr). Esaxerenone was initiated at 1.25 mg/day and followed by incremental dose escalation based on BP and serum potassium level monitoring. The treatment period was 12 weeks. The primary endpoint was change in morning home systolic BP/diastolic BP (SBP/DBP) from baseline to end of treatment (EOT). Secondary endpoints included achievement rate of target BP, change in UACR from baseline, and safety. RESULTS: In total, 113 patients were enrolled. Morning home SBP/DBP significantly decreased from baseline to EOT in the total population (- 11.6/- 5.2 mmHg, both p < 0.001) and in all UACR subcohorts (all p < 0.001). The target BP achievement rate was 38.5%. Significant reductions in bedtime home and office BPs were also shown in the total population and all UACR subcohorts. UACR significantly improved from baseline to EOT in the total (- 50.9%, p < 0.001) and all UACR subcohorts (all p < 0.001). Incidence of serum potassium elevation as drug-related treatment emergent adverse events was 2.7%. The change from baseline in estimated glomerular filtration rate (eGFR) was - 4.8 mL/min/1.73 m2. CONCLUSION: Esaxerenone demonstrated a BP-lowering effect and improved albuminuria. The effects were consistent regardless of the severity of albuminuria without clinically relevant serum potassium elevation and eGFR reduction. CLINICAL TRIAL REGISTRATION: jRCTs06119002.
Subject(s)
Diabetes Mellitus , Diabetic Nephropathies , Hypertension , Albumins/therapeutic use , Albuminuria/drug therapy , Albuminuria/etiology , Blood Pressure , Creatinine/pharmacology , Creatinine/therapeutic use , Diabetic Nephropathies/complications , Diabetic Nephropathies/drug therapy , Glomerular Filtration Rate , Humans , Hypertension/complications , Hypertension/drug therapy , Potassium/pharmacology , Potassium/therapeutic use , Prospective Studies , Pyrroles , SulfonesABSTRACT
Head Injury Assessment (HIA) is the screening tool for head injury during a rugby game. The purpose of this study was to investigate the epidemiology of HIA in the Japan Rugby Top League (JRTL). The incidences of HIA, defined concussion (per 1,000 player-hours) and repeated concussions were evaluated in three seasons (2016-17, 2017-18, 2018-19; total 360 games). The HIA incidence rates were 12.7 (95% confidence interval 9.5-15.9), 20.8 (16.8-24.9), and 25.0 (20.5-29.5) in each season. HIA-1 criteria 2, which is applied for suspected concussion cases, was performed for 46 cases in the 2016-17 season, 81 cases in the 2017-18 season, and 88 cases in the 2018-19 season. The concussion incidence rates were significantly greater in the 2017-18 season (9.6/1000 player-hours, 95% confidence interval 6.8-12.4) and the 2018-19 season (14.4, 11-17.8) compared to the 2016-17 season (4.8, 2.8-6.8). The number of repeated concussion cases in the same season was 1 in the 2016-17 season and 4 in both the 2017-18 and 2018-19 seasons. This study confirmed significantly higher HIA and concussion incidence rates over time. Although the HIA system might have been established in the three seasons in JRTL, comprehensive management needs to be improved to prevent repeated concussions.
Subject(s)
Athletic Injuries , Brain Concussion , Craniocerebral Trauma , Football , Athletic Injuries/diagnosis , Brain Concussion/diagnosis , Craniocerebral Trauma/epidemiology , Football/injuries , Humans , Incidence , Japan/epidemiology , Rugby , SeasonsABSTRACT
AIM: To evaluate the effect of canagliflozin, a sodium-glucose co-transporter-2 (SGLT2) inhibitor, on albuminuria and the decline of estimated glomerular filtration rate (eGFR) in participants with type 2 diabetes and microalbuminuria. METHODS: The CANPIONE study is a multicentre, randomized, parallel-group and open-labelled study consisting of a unique 24-week preintervention period, during which the rate of eGFR decline before intervention is estimated, followed by a 52-week intervention and a 4-week washout period. Participants with a geometric mean urinary albumin-to-creatinine ratio (UACR) of 50 and higher and less than 300 mg/g in two consecutive first-morning voids at two different time points, and an eGFR of 45 ml/min/1.73m2 or higher, are randomly assigned to receive canagliflozin 100 mg daily or to continue guideline-recommended treatment, except for SGLT2 inhibitors. The first primary outcome is the change in UACR, and the second primary outcome is the change in eGFR slope. RESULTS: A total of 258 participants were screened and 98 were randomized at 21 sites in Japan from August 2018 to May 2021. The mean baseline age was 61.4 years and 25.8% were female. The mean HbA1c was 7.9%, mean eGFR was 74.1 ml/min/1.73m2 and median UACR was 104.2 mg/g. CONCLUSIONS: The CANPIONE study will determine whether the SGLT2 inhibitor canagliflozin can reduce albuminuria and slow eGFR decline in participants with type 2 diabetes and microalbuminuria.
Subject(s)
Diabetes Mellitus, Type 2 , Sodium-Glucose Transporter 2 Inhibitors , Albuminuria/epidemiology , Canagliflozin/therapeutic use , Diabetes Mellitus, Type 2/complications , Diabetes Mellitus, Type 2/drug therapy , Diabetes Mellitus, Type 2/urine , Female , Glomerular Filtration Rate , Humans , Japan/epidemiology , Male , Middle Aged , Sodium-Glucose Transporter 2 Inhibitors/pharmacology , Sodium-Glucose Transporter 2 Inhibitors/therapeutic useABSTRACT
Cellobiohydrolase I from Trichoderma reesei ( Tr Cel7A) is one of the best-studied cellulases, exhibiting high activity towards crystalline cellulose. Tryptophan residues at subsites -7 and -4 (Trp40 and Trp38 respectively) are located at the entrance and middle of the tunnel-like active site of Tr Cel7A, and are conserved among the GH family 7 cellobiohydrolases. Trp40 of Tr Cel7A is important for the recruitment of cellulose chain ends on the substrate surface, but the role of Trp38 is less clear. Comparison of the effects of W38A and W40A mutations on the binding energies of sugar units at the two subsites indicated that the contribution of Trp38 to the binding was greater than that of Trp40. In addition, the smooth gradient of binding energy was broken in W38A mutant. To clarify the importance of Trp38, the activities of Tr Cel7A WT and W38A towards crystalline cellulose and amorphous cellulose were compared. W38A was more active than WT towards amorphous cellulose, whereas its activity towards crystalline cellulose was only one-tenth of that of WT. To quantify the effect of mutation at subsite -4, we measured kinetic parameters of Tr Cel7A WT, W40A and W38A towards cello-oligosaccharides. All combinations of enzymes and substrates showed substrate inhibition, and comparison of the inhibition constants showed that the Trp38 residue increases the velocity of substrate intake ( k on for forming productive complex) from the minus side of the subsites. These results indicate a key role of Trp38 residue in processively loading the reducing-end of cellulose chain into the catalytic tunnel.
ABSTRACT
We demonstrate a method for label-free monitoring of hydrolytic activity of crystalline-chitin-degrading enzyme, chitinase, by means of Raman spectroscopy. We found that crystalline chitin exhibited a characteristic Raman peak at 2995 cm-1, which did not appear in the reaction product, N,N'-diacetylchitobiose. We used this Raman peak as a marker of crystalline chitin degradation to monitor the hydrolytic activity of chitinase. When the crystalline chitin suspension and chitinase were mixed together, the peak intensity of crystalline chitin at 2995 cm-1 was linearly decreased depending on incubation time. The decrease in peak intensity was inversely correlated with the increase in the amount of released N,N'-diacetylchitobiose, which was measured by conventional colorimetric assay with alkaline ferricyanide. Our result, presented here, provides a new method for simple, in situ, and label-free monitoring of enzymatic activity of chitinase.
Subject(s)
Chitinases , Chitin , Hydrolysis , Spectrum Analysis, Raman , SuspensionsABSTRACT
OBJECTIVES: Acute subdural hematoma (ASDH) is known to be devasting sport-related head injury but it is relatively rare in rugby compared with other contact sports. Certain cases of ASDH have happened in high school rugby players in Japan. To prevent them from the injury we report a background of the players. METHODS: Data of high school rugby players who suffered ASDH were extracted from injury reports in the Japan Rugby Football Union between April 2004 and March 2020. The number of injured players, diagnosis on the report, school year, phase of play where the injury occurred, and playing career were analyzed. RESULTS: There were 30 cases of ASDH including 16 cases in the first year, 9 in the second year, and 5 in the third year of playing. Phase of play was mainly being tackled in 11 (37%), and tackling in 13 (43%). Novice players, defined as a player having less playing experience of rugby during junior high school, accounted for 77% of phase of tackling, 82% of being tackled. First year novice players accounted for 100% of phase of being tackled. Outcome within 6 months after injury was recovery in 14, morbidity in 6, mortality in 2, and unknown in 8. CONCLUSIONS: Playing experience in high school rugby players should be considered as an important factor for prevention of ASDH-in particular, phase of being tackled is riskier than that of tackling for first year novice players.
Subject(s)
Craniocerebral Trauma/epidemiology , Football/injuries , Football/trends , Hematoma, Subdural, Acute/epidemiology , Schools/trends , Adolescent , Athletic Injuries/diagnosis , Athletic Injuries/epidemiology , Cohort Studies , Craniocerebral Trauma/diagnosis , Craniocerebral Trauma/prevention & control , Female , Hematoma, Subdural, Acute/diagnosis , Hematoma, Subdural, Acute/prevention & control , Humans , Japan/epidemiology , Male , Retrospective StudiesABSTRACT
Pontin and Reptin are closely related proteins belonging to the AAA+ (ATPases Associated with various cellular Activities) family. They form a hetero-oligomeric complex, Pontin/Reptin, which is involved in protein stability and assembly of the protein complexes as a molecular chaperone. Overexpression of Pontin and Reptin in tumor cells has been reported and is implicated in the development of various cancers. However, the molecular mechanism of Pontin/Reptin function in oral squamous cell carcinoma (OSCC) development remains unclear. Here, we identify HEAT repeat-containing protein 1 (HEATR1) as a novel binding factor of Pontin/Reptin. Functionally, HEATR1 stabilizes Pontin/Reptin and positively regulates OSCC cell proliferation by activating mTOR and pre-rRNA synthesis. We also find that HEATR1 expression is markedly upregulated in tumor region of OSCC tissue. Hence, we propose that HEATR1 is involved in the regulation of mTOR and ribosome biogenesis as a potential protein stabilizer of Pontin/Reptin in OSCC.
Subject(s)
ATPases Associated with Diverse Cellular Activities/metabolism , Carrier Proteins/metabolism , Cell Proliferation/genetics , DNA Helicases/metabolism , Minor Histocompatibility Antigens/metabolism , Mouth Neoplasms/metabolism , RNA-Binding Proteins/metabolism , Squamous Cell Carcinoma of Head and Neck/metabolism , ATPases Associated with Diverse Cellular Activities/genetics , Carrier Proteins/genetics , Cell Line, Tumor , DNA Helicases/genetics , Gene Expression Regulation, Neoplastic/genetics , Genes, rRNA , Humans , Immunohistochemistry , Minor Histocompatibility Antigens/genetics , Mouth Neoplasms/genetics , Protein Binding , RNA Interference , RNA-Binding Proteins/genetics , Real-Time Polymerase Chain Reaction , Squamous Cell Carcinoma of Head and Neck/genetics , TOR Serine-Threonine Kinases/metabolism , Tandem Mass Spectrometry , Up-RegulationABSTRACT
BACKGROUND: The Rugby World Cup (RWC) is one of the biggest international mega sports events in the world. This study was conducted to identify and evaluate the volume, nature, and severity of spectator medical care in the stadiums of 12 venues across Japan during RWC 2019. METHOD: This was a retrospective review of medical records from spectator medical rooms of 45 official matches of RWC 2019 between September 20 and November 2, 2019. All patients in the stadium who visited the spectator medical room and were transferred to a hospital were included. The wet bulb globe temperature (WBGT) value at the kick-off time of each match, the number of visits to the spectator medical room, and the number of transfers to a hospital were reviewed and analyzed. The patient presentation rate (PPR) was calculated per 10,000 attendees. Severity categories were defined as mild or severe. Mild cases were considered non-life threatening requiring minimal medical intervention, and severe cases required transport to a hospital. RESULT: The total number of visits to the spectator medical room was 449 with a PPR of 2.63. Most cases (91.5%) were mild in severity. The PPR was significantly higher for the matches held with a WBGT over 25 °C than for the matches under 21 °C (PPR 4.27 vs 2.04, p = 0.04). Thirty-eight cases were transferred to a hospital by ambulance; the PPR was 0.22. The most common reasons for transfer to the hospital were heat illness and fracture/dislocation, at a rate of 15.8% each. The incidence rate of cardiopulmonary arrest per 10,000 attendees was 0.0059 during RWC 2019. CONCLUSION: Preparation and provision of appropriate medical service for spectators is a key factor for mass-gathering events. During RWC 2019, the majority (91.5%) of patients who sought medical attention did so for minor complaints, which were easily assessed and managed. On the other hand, a higher WBGT situation contributes significantly to an increased PPR (< 21 versus > 25, 2.04 versus 4.27, p = 0.04). Careful medical preparation, management, and development of public education programs for higher WBGT situations will be required in the future for similar international mega sports events.
Subject(s)
Ambulatory Care/statistics & numerical data , Emergency Service, Hospital/statistics & numerical data , Hospitalization/statistics & numerical data , Mass Behavior , Adolescent , Adult , Aged , Aged, 80 and over , Anniversaries and Special Events , Child , Child, Preschool , Female , Football , Humans , Infant , Infant, Newborn , Japan , Male , Middle Aged , Retrospective Studies , Young AdultABSTRACT
Motor proteins are essential units of life and are well-designed nanomachines working under thermal fluctuations. These proteins control moving direction by consuming chemical energy or by dissipating electrochemical potentials. Chitinase A from bacterium Serratia marcescens (SmChiA) processively moves along crystalline chitin by hydrolysis of a single polymer chain to soluble chitobiose. Recently, we directly observed the stepping motions of SmChiA labeled with a gold nanoparticle by dark-field scattering imaging to investigate the moving mechanism. Time constants analysis revealed that SmChiA moves back and forth along the chain freely, because forward and backward states have a similar free energy level. The similar probabilities of forward-step events (83.5%=69.3%+14.2%) from distributions of step sizes and chain-hydrolysis (86.3%=(1/2.9)/(1/2.9+1/18.3)×100) calculated from the ratios of time constants of hydrolysis and the backward step indicated that SmChiA moves forward as a result of shortening of the chain by a chitobiose unit, which stabilizes the backward state. Furthermore, X-ray crystal structures of sliding intermediate and molecular dynamics simulations showed that SmChiA slides forward and backward under thermal fluctuation without large conformational changes of the protein. Our results demonstrate that SmChiA is a burnt-bridge Brownian ratchet motor.
ABSTRACT
Serratia marcescens chitinase A (SmChiA) processively hydrolyzes recalcitrant biomass crystalline chitin under mild conditions. Here, we combined multiple sequence alignment, site-saturation mutagenesis, and automated protein purification and activity measurement with liquid-handling robot to reduce the number of mutation trials and shorten the screening time for hydrolytic activity improvement of SmChiA. The amino acid residues, which are not conserved in the alignment and are close to the aromatic residues along the substrate-binding sites in the crystal structure, were selected for site-saturation mutagenesis. Using the previously identified highly active F232W/F396W mutant as a template, we identified the F232W/F396W/S538V mutant, which shows further improved hydrolytic activity just by trying eight different sites. Importantly, valine was not found in the multiple sequence alignment at Ser538 site of SmChiA. Our combined approach allows engineering of highly active enzyme mutants, which cannot be identified only by the introduction of predominant amino acid residues in the multiple sequence alignment.
ABSTRACT
Cellobiohydrolases directly convert crystalline cellulose into cellobiose and are of biotechnological interest to achieve efficient biomass utilization. As a result, much research in the field has focused on identifying cellobiohydrolases that are very fast. Cellobiohydrolase A from the bacterium Cellulomonas fimi (CfCel6B) and cellobiohydrolase II from the fungus Trichoderma reesei (TrCel6A) have similar catalytic domains (CDs) and show similar hydrolytic activity. However, TrCel6A and CfCel6B have different cellulose-binding domains (CBDs) and linkers: TrCel6A has a glycosylated peptide linker, whereas CfCel6B's linker consists of three fibronectin type 3 domains. We previously found that TrCel6A's linker plays an important role in increasing the binding rate constant to crystalline cellulose. However, it was not clear whether CfCel6B's linker has similar function. Here we analyze kinetic parameters of CfCel6B using single-molecule fluorescence imaging to compare CfCel6B and TrCel6A. We find that CBD is important for initial binding of CfCel6B, but the contribution of the linker to the binding rate constant or to the dissociation rate constant is minor. The crystal structure of the CfCel6B CD showed longer loops at the entrance and exit of the substrate-binding tunnel compared with TrCel6A CD, which results in higher processivity. Furthermore, CfCel6B CD showed not only fast surface diffusion but also slow processive movement, which is not observed in TrCel6A CD. Combined with the results of a phylogenetic tree analysis, we propose that bacterial cellobiohydrolases are designed to degrade crystalline cellulose using high-affinity CBD and high-processivity CD.
Subject(s)
Bacterial Proteins/chemistry , Cellulomonas/enzymology , Cellulose 1,4-beta-Cellobiosidase/chemistry , Fungal Proteins/chemistry , Hypocreales/enzymology , Bacterial Proteins/metabolism , Binding Sites , Catalytic Domain , Cellulomonas/chemistry , Cellulomonas/metabolism , Cellulose/metabolism , Cellulose 1,4-beta-Cellobiosidase/metabolism , Crystallography, X-Ray , Fungal Proteins/metabolism , Hypocreales/chemistry , Hypocreales/metabolism , Models, Molecular , Protein Binding , Protein Conformation , Protein Domains , Substrate SpecificityABSTRACT
Cellulose is the most abundant biomass on Earth, and many microorganisms depend on it as a source of energy. It consists mainly of crystalline and amorphous regions, and natural degradation of the crystalline part is highly dependent on the degree of processivity of the degrading enzymes (i.e., the extent of continuous hydrolysis without detachment from the substrate cellulose). Here, we report high-speed atomic force microscopic (HS-AFM) observations of the movement of four types of cellulases derived from the cellulolytic bacteria Cellulomonas fimi on various insoluble cellulose substrates. The HS-AFM images clearly demonstrated that two of them (CfCel6B and CfCel48A) slide on crystalline cellulose. The direction of processive movement of CfCel6B is from the nonreducing to the reducing end of the substrate, which is opposite that of processive cellulase Cel7A of the fungus Trichoderma reesei (TrCel7A), whose movement was first observed by this technique, while CfCel48A moves in the same direction as TrCel7A. When CfCel6B and TrCel7A were mixed on the same substrate, "traffic accidents" were observed, in which the two cellulases blocked each other's progress. The processivity of CfCel6B was similar to those of fungal family 7 cellulases but considerably higher than those of fungal family 6 cellulases. The results indicate that bacteria utilize family 6 cellulases as high-processivity enzymes for efficient degradation of crystalline cellulose, whereas family 7 enzymes have the same function in fungi. This is consistent with the idea of convergent evolution of processive cellulases in fungi and bacteria to achieve similar functionality using different protein foldings.
Subject(s)
Bacterial Proteins/chemistry , Cellulases/chemistry , Cellulomonas/enzymology , Fungal Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Biocatalysis , Biological Evolution , Cellulases/genetics , Cellulases/metabolism , Cellulomonas/chemistry , Cellulomonas/genetics , Cellulomonas/metabolism , Cellulose/chemistry , Cellulose/metabolism , Fungal Proteins/genetics , Fungal Proteins/metabolism , Kinetics , Microscopy, Atomic ForceABSTRACT
This study aimed to clarify the cause of rugby head and spinal cord injuries through a network centrality analysis of 14-year (2004-2018) longitudinal data in Japan. The study hypothesis is that understanding the causal relationship among the occurrence of serious injuries, the quality of player experience and play situation as a network structure could be possible to obtain practical knowledge on injury prevention. In this study, bipartite graphs are used to make it easier to understand the situation of players and injuries. This would also help to elucidate more characteristic subgroup. A network bipartite graph and subgroup (cluster) analyses were performed to clarify the injured players' experience and the cause of injury. We used the algorithm of R program, IGRAPH, clustering edge betweenness. For subgroup extraction, the modularity Q value was used to determine which step to cut. The Japanese rugby population was 93,873 (2014-2018 average), and 27% were high school students. The data showed that careful attention would be particularly needed for groups of inexperienced Japanese high school players. Our study suggests that we should consider introducing rules that prohibit "head-on collisions" in youth rugby.