High Hydrolytic Activity Research Articles

High hydrolytic activity of the metagenomic lipase LipC12 in deep eutectic solvents

Deep eutectic solvents (DESs) have been shown to increase the activity of lipases in biocatalytic processes, especially when added at intermediate concentrations. We evaluated the effect of DESs on the hydrolysis of p-nitrophenyl palmitate (p-NPP) by a metagenomic lipase, LipC12. The hydrogen bond acceptor was choline chloride (ChCl), while sorbitol (Sor), xylitol (Xyl), Glycerol (Gly), ethylene glycol (EG), triethylene glycol (TEG) and urea (U) were tested as hydrogen bond donors. The best results were obtained at 25% (v v−1) DES, with the increases in hydrolytic activity, compared to activity in buffer, being 18-fold for ChCl:Sor (2:1), and 10- to 12-fold for ChCl:Gly (1:2), ChCl:EG (1:2) and ChCl:Xyl (2:1). Dynamic Light Scattering analysis suggests that protein disaggregation played a key role in this activation: the hydrodynamic radius of LipC12 was 157 nm in buffer, but ranged from about 1 to 7 nm in DESs at different concentrations. The addition of 100 mM NaCl to the DES-containing media (at 25% v v−1 DES) slightly improved LipC12 activity. Addition of the hydrogen-bond donor alone also increased the p-NPP hydrolyzing activity with 30% (w v−1) sorbitol or xylitol giving 4.5-fold higher activities, compared to activity in buffer. Our fold-improvements in activity are the highest yet reported for the p-NPP-hydrolyzing activity of lipases and suggest that more studies are required, especially with other biocatalytic reactions such as esterification and transesterification.

Molecular mechanism for endo-type action of glycoside hydrolase family 55 endo-β-1,3-glucanase on β1-3/1-6-glucan

The glycoside hydrolase family 55 (GH55) includes inverting exo-β-1,3-glucosidases and endo-β-1,3-glucanases, acting on laminarin, which is a β1-3/1-6-glucan consisting of a β1-3/1-6-linked main chain and β1-6-linked branches. Despite their different modes of action toward laminarin, endo-β-1,3-glucanases share with exo-β-1,3-glucosidases conserved residues that form the dead-end structure of subsite-1. Here, we investigated the mechanism of endo-type action on laminarin by GH55 endo-β-1,3-glucanase MnLam55A, identified from Microdochium nivale. MnLam55A, like other endo-β-1,3-glucanases, degraded internal β-d-glucosidic linkages of laminarin, producing more reducing sugars than the sum of d-glucose and gentiooligosaccharides detected. β1-3-Glucans lacking β1-6-linkages in the main chain were not hydrolyzed. NMR analysis of the initial degradation of laminarin revealed that MnLam55A preferentially cleaved the nonreducing terminal β1-3-linkage of the laminarioligosaccharide moiety at thereducing end side of the main chain β1-6-linkage. MnLam55A liberates d-glucose from laminaritriose and longer laminarioligosaccharides, but kcat/Km values to laminarioligosaccharides (≤4.21s-1mM-1) were much lower than to laminarin (5920s-1mM-1). These results indicate that β-glucan binding to the minus subsites of MnLam55A, including exclusive binding of the gentiobiosyl moiety to subsites-1 and-2, is required for high hydrolytic activity. A crystal structure of MnLam55A, determined at 2.4 Å resolution, showed that MnLam55A adopts an overall structure and catalytic site similar to those of exo-β-1,3-glucosidases. However, MnLam55A possesses an extended substrate-binding cleft that is expected to form the minus subsites. Sequence comparison suggested that other endo-type enzymes share the extended cleft. The specific hydrolysis of internal linkages in laminarin is presumably common to GH55 endo-β-1,3-glucanases.

Hydrolase mimic via second coordination sphere engineering in metal-organic frameworks for environmental remediation

Enzymes achieve high catalytic activity with their elaborate arrangements of amino acid residues in confined optimized spaces. Nevertheless, when exposed to complicated environmental implementation scenarios, including high acidity, organic solvent and high ionic strength, enzymes exhibit low operational stability and poor activity. Here, we report a metal-organic frameworks (MOFs)-based artificial enzyme system via second coordination sphere engineering to achieve high hydrolytic activity under mild conditions. Experiments and theoretical calculations reveal that amide cleavage catalyzed by MOFs follows two distinct catalytic mechanisms, Lewis acid- and hydrogen bonding-mediated hydrolytic processes. The hydrogen bond formed in the secondary coordination sphere exhibits 11-fold higher hydrolytic activity than the Lewis acidic zinc ions. The MOFs exhibit satisfactory degradation performance of toxins and high stability under extreme working conditions, including complicated fermentation broth and high ethanol environments, and display broad substrate specificity. These findings hold great promise for designing artificial enzymes for environmental remediation.

Tuning surface interactions on MgFe2O4 nanoparticles to induce interfacial hyperactivation in Candida rugosa lipase immobilization

Lipase adsorption on solid supports can be mediated by a precise balance of electrostatic and hydrophobic interactions. A suitable fine-tuning could allow the immobilized enzyme to display high catalytic activity. The objective of this work was to investigate how pH and ionic strength fluctuations affected protein-support interactions during immobilization via physical adsorption of a Candida rugosa lipase (CRL) on MgFe2O5. The highest amount of immobilized protein (IP) was measured at pH 4, and an ionic strength of 90 mM. However, these immobilization conditions did not register the highest hydrolytic activity (HA) in the biocatalyst (CRLa@MgFe2O4), finding the best values also at acidic pH but with a slight shift towards higher values of ionic strength around 110 mM. These findings were confirmed when the adsorption isotherms were examined under different immobilization conditions so that the maximum measurements of IP did not coincide with that of HA. Furthermore, when the recovered activity was examined, a strong interfacial hyperactivation of the lipase was detected towards acidic pH and highly charged surrounding environments. Spectroscopic studies, as well as in silico molecular docking analyses, revealed a considerable involvement of surface hydrophobic protein-carrier interactions, with aromatic aminoacids, especially phenylalanine residues, playing an important role. In light of these findings, this study significantly contributes to the body of knowledge and a better understanding of the factors that influence the lipase immobilization process on magnetic inorganic oxide nanoparticle surfaces.

Three-directional engineering of IsPETase with enhanced protein yield, activity, and durability

The mesophilic PETase from Ideonella sakaiensis (IsPETase) has been shown to exhibit high PET hydrolysis activity, but its low stability limits its industrial applications. Here, we developed a variant, Z1-PETase, with enhanced soluble protein yield and durability while maintaining or improving activity at lower temperatures. The selected Z1-PETase not only exhibited a 20-fold improvement in soluble protein yield compared to the previously engineered IsPETaseS121E/D186H/S242T/N246D (4p) variant, but also demonstrated a 30% increase in low-temperature activity at 40 °C, along with an 11 °C increase in its TmD value. The PET depolymerization test across a temperature range low to high (30–70 °C) confirmed that Z1-PETase exhibits high accessibility of mesophilic PET hydrolase and rapid depolymerizing rate at higher temperature in accordance with the thermal behaviors of polymer and enzyme. Additionally, structural interpretation indicated that the stabilization of specific active site loops in Z1-PETase contributes to enhanced thermostability without adversely impacting enzymatic activity. In a pH-stat bioreactor, Z1-PETase depolymerized > 90% of both transparent and colored post-consumer PET powders within 24 and 8 h at 40 °C and 55 °C, respectively, demonstrating that the utility of this IsPETase variant in the bio-recycling of PET.

Targeted metabolic profiling of the revived ancient ‘Corbella’ olive cultivar during early maturation

‘Corbella’ is an ancient olive cultivar whose cultivation has recently been revived and hence little is known about its composition. This is the first work studying the metabolic profile of ‘Corbella’ olives during early maturation. Olives with a ripening index (RI) < 1 yielded considerably less oil content (<40%) but had more concentration of phenolic compounds (148.41–219.70 mg/kg), carotenoids (9.61–14.94 mg/kg) and squalene (521.41–624.40 mg/kg). Contrarily, the levels of α-tocopherol were higher at the RI of 1.08 and 1.96 (64.57 and 57.75 mg/kg, respectively). The most abundant phenolic compound was oleuropein aglycone (>50% of the phenolic composition), suggesting a high hydrolytic activity of β-glucosidase in the fruit. The antioxidant capacity was barely affected, while oleic/linoleic ratio reached its highest at RI of 1.96. Therefore, olives with an RI below 2 could be good candidates to produce high-quality olive oils with good level of stability.

Design of SC PEP with enhanced stability against pepsin digestion and increased activity by machine learning and structural parameters modeling

Prolyl endopeptidases from Sphingomonas capsulata (SC PEP) has attracted much attention as promising oral therapy candidate for celiac sprue, however, its low stability in the gastric environment leads to unsatisfactory clinical results. Therefore, improving its stability against pepsin digestion at low pH is crucial for clinical applications, but challenging. In this study, machine learning and physical parameter model were combined to design SC PEP mutants. After iterations, 20 mutants had higher hydrolysis activity in stomach environment, which was up to 14.1-fold compared with wild-type SC PEP. Mutant M24 involving stable and active mutations and pegylated M24 (M24-PEG) had higher activity of hydrolyzing immunogen in bread than wild-type SC PEP in vitro and in vivo, and residual immunogens in simulated gastric environment were only 1/8 and 1/10 of that in the wild-type SC PEP group. The total residual immunogens in the gastrointestinal tract of mice in the M24 and M24-PEG groups were <20 ppm, reaching the standard of non-toxic food. Our results indicate that the combination of M24 (or M24-PEG) with EP-B2 may be a promising candidate for celiac disease, and the strategies developed in this study provide a paradigm for the design of SC PEP stability mutants.

Improving the cellobiose hydrolysis activity of glucose-stimulating β-glucosidase Bgl2A

β-Glucosidases with high catalytic activity and glucose tolerant properties possess promising applications in lignocellulose-based industries. To obtain enzymes possessing these properties, a semi-rational strategy was employed to engineer the glucose-stimulating β-glucosidase Bgl2A for high cellobiose hydrolysis activity. A total of 18 mutants were constructed. A22S, V224D, and A22S/V224D exhibited high specific activities of 272.06, 237.60, and 239.29 U/mg toward cellobiose, which were 2.5- to 2.8-fold of Bgl2A. A22S, V224D, and A22S/V224D exhibited increased kcat values, which were 2.7- to 3.1-fold of Bgl2A. A22S and V224D maintained glucose-stimulating property, whereas A22S/V224D lost it. Using 150 g/L cellobiose as the substrate, the amount of glucose produced by A22S was the highest, yielding 129.70 g/L glucose after 3 h reaction at 35 °C. The synergistic effects of the engineered enzymes with commercial cellulase on hydrolyzing cellulose were investigated. Supplemented with the commercial cellulase and A22S, the highest glucose amount of 23.30 g/L was yielded from cellulose with hydrolysis rate of 21.02 %. Given its high cellobiose hydrolysis activity and glucose-stimulating properties, A22S can be used as a component of enzyme cocktail to match mesophilic cellulases for efficient cellulose hydrolysis.

Enhanced enzyme thermostability of a family I.3 lipase LipSR1 by T118A mutation at the calcium-binding site.

The lipase gene lipSR1 isolated from oil-contaminated soil exhibits high hydrolytic activity for short-chain fatty acid substrates. A single calcium ion is required to anchor the lid of LipSR1 in an open conformation by coordination with two aspartate residues and three other residues in the lid. The lid of LipSR1 is anchored by Ca2+, which is coordinated by side-chain carboxyl oxygens of Asp153 and Asp157, carbonyl oxygens of Thr118 and Ser144, and the side chain of Gln120. D157A, D153R, Q120A, S144A, and T118A mutants were produced by site-directed mutagenesis in this study. Analyses of hydrolytic activity and thermostability showed that the properties of D157A, D153R, Q120A, and S144A were almost lost, suggesting that Asp157, Asp153, Gln120, and Ser144 are important residues for LipSR1. However, the catalytic performance of T118A was clearly maintained. Moreover, the thermostability of mutant T118A was higher than that of wild-type LipSR1. These results indicated that mutation of threonine at position 118 improved the stability of the enzyme at high temperature.

Untargeted metabolomic profiling of Aspergillus sojae 3.495 and Aspergillus oryzae 3.042 fermented soy sauce koji and effect on moromi fermentation flavor

Aspergillus sojae 3.495 and Aspergillus oryzae 3.042 are essential microbial starters in fermented foods due to their high hydrolytic enzyme activities. However, their metabolic characteristics and differences during soy sauce fermentation are still unclear. This study identified 42 non-volatile differential metabolites based on UHPLC-MS/MS, among which 10 amino acids and derivatives and 4 non-volatile acids were high in A. sojae 3.495 koji, while 3 sugar alcohols, 5 flavonoids, and 4 fatty acids were prominent in A. oryzae 3.042. Further analysis revealed that higher acidic protease (1887.43 U/g) in A. sojae 3.495 and higher glucoamylase (2413.36 U/g) and cellulase (890.78 U/g) in A. oryzae 3.042 dominated the formation of differential metabolites. Metabolic pathway analysis showed that shikimate and phenylalanine metabolic pathways in A. sojae 3.495 were more active and enhanced 3-methyl-butanal and 2-phenylethanal in moromi fermentation. The more prominent pathways of sugar alcohol metabolism and flavonoid biosynthesis in A. oryzae 3.042 koji enhanced 2-methyl-butanoic acid and ethyl 3-methylbutyrate in moromi and significantly elevated reducing sugars (1.56 g/100 mL). Overall, comparative metabolomics elucidated the metabolic framework and flavor formation mechanisms of different inocula in soy sauce koji, providing ideas for scientific control of soy sauce fermentation and multi-strain koji-making for flavor enhancement.

Biosynthesis of the acid protease produced by Lacticaseibacillus casei LBC 237 and Limosilactobacillus fermentum LBF 433 and their potential application in the bovine milk clotting

This study aimed to produce and evaluate proteases from Lacticaseibacillus casei (LBC 237) and Limosilactobacillus fermentum (LBF 433) for milk coagulation. Proteases were assessed for acid protease activity (PA) and stability within temperature range of 30–75 °C and pH levels of 5.0, 5.5, and 6.8. Substrate concentration (0.025 %–2.5 %) and storage conditions (up to 120 days) were also investigated. Milk-clotting activity (MCA) and the effect of NaCl (1–4 %) on coagulation were evaluated, with subsequent characterization of resulting clots in terms of molecular size, structure, and flavor profile. Chemical changes in fermentation, proteases, and clots were determined. The proteases showed better PA at 60 °C and a pH of 5.5, with no change over 120 days. LBC-P demonstrated a higher affinity for the substrate. The proteases generated bands at ∼35 kDa, similar to bovine chymosin bands, and LBC-P exhibited MCA (480 SU/mL). Clots formed using LBC-P and bovine chymosin displayed similar bands (50 kDa, 35 kDa, and 15 kDa), while the LBC-P clot exhibited a more porous structure. LBC-P was inactivated by NaCl concentrations (≥1 %), and the clots showed changes in amides I. The bitterness intensity was lower for the clot obtained with LBC-P. While both LBC-P and LBF-P showed potential for protein hydrolysis, only LBC-P demonstrated potential for milk coagulation for cheese production. This study suggests acid proteases with high hydrolytic activity. In conclusion, L. casei (LBC 237) derived proteases show potential for milk coagulation, offering a promising alternative to bovine chymosin in yellow cheese production.

Function and Structure of Lacticaseibacillus casei GH35 β-Galactosidase LBCZ_0230 with High Hydrolytic Activity to Lacto-N-biose I and Galacto-N-biose.

β-Galactosidase (EC 3.2.1.23) hydrolyzes β-D-galactosidic linkages at the non-reducing end of substrates to produce β-D-galactose. Lacticaseibacillus casei is one of the most widely utilized probiotic species of lactobacilli. It possesses a putative β-galactosidase belonging to glycoside hydrolase family 35 (GH35). This enzyme is encoded by the gene included in the gene cluster for utilization of lacto-N-biose I (LNB; Galβ1-3GlcNAc) and galacto-N-biose (GNB; Galβ1-3GalNAc) via the phosphoenolpyruvate: sugar phosphotransferase system. The GH35 protein (GnbG) from L. casei BL23 is predicted to be 6-phospho-β-galactosidase (EC 3.2.1.85). However, its 6-phospho-β-galactosidase activity has not yet been examined, whereas its hydrolytic activity against LNB and GNB has been demonstrated. In this study, L. casei JCM1134 LBCZ_0230, homologous to GnbG, was characterized enzymatically and structurally. A recombinant LBCZ_0230, produced in Escherichia coli, exhibited high hydrolytic activity toward o-nitrophenyl β-D-galactopyranoside, p-nitrophenyl β-D-galactopyranoside, LNB, and GNB, but not toward o-nitrophenyl 6-phospho-β-D-galactopyranoside. Crystal structure analysis indicates that the structure of subsite -1 of LBCZ_0230 is very similar to that of Streptococcus pneumoniae β-galactosidase BgaC and not suitable for binding to 6-phospho-β-D-galactopyranoside. These biochemical and structural analyses indicate that LBCZ_0230 is a β-galactosidase. According to the prediction of LNB's binding mode, aromatic residues, Trp190, Trp240, Trp243, Phe244, and Tyr458, form hydrophobic interactions with N-acetyl-D-glucosamine residue of LNB at subsite +1.

Dry-to-wet fluctuation of moisture contents enhanced the mineralization of chloramphenicol antibiotic

Due to livestock wastewater irrigation, soil is becoming one of the major sinks of antibiotics in the environment. Recently, it is getting recognized that a variety of minerals under low moisture conditions can induce strong catalytic hydrolysis to antibiotics. However, the relative importance and implication of soil water content (WC) for natural attenuation of soil residual antibiotics has not been well recognized. In order to explore the optimal moisture levels and the key soil properties dominating for the high catalytic hydrolysis activities of soils, this study collected 16 representative soil samples across China, and assessed their performances to degrade chloramphenicol (CAP) under different moisture levels. The results showed that the soils with low organic matter contents (< 20 g/kg) and high amounts of crystalline Fe/Al were particularly effective in catalyzing CAP hydrolysis when exposed to low WC (< 6%, wt/wt), leading to CAP hydrolysis half-lives of <40 d Higher WC greatly suppressed the catalytic activity of the soil. By utilizing this process, it is possible to integrate abiotic and biotic degradation to enhance the mineralization of CAP, attributing to that the hydrolytic products are more available for soil microorganisms. As expected, the soils experienced periodic dry-to-wet moisture conditions (i.e., the WC shifting from 1 to 5% to 20–35%, wt/wt) exhibited higher degradation and mineralization of 14C-CAP, in comparison with the constant wet treatment. Meanwhile, the bacterial community composition and the specific genera showed that the dry-to-wet fluctuation of soil WC relieved the antimicrobial stress to bacterial community. Our study verifies the critical role of soil WC in mediating the natural attenuation of antibiotics, and guides to remove antibiotics from both wastewater and soil.

Modification of the properties of the metagenomic lipase LipC12 by engineering of the hydrophobic cavity

Kinetic resolution of racemates with lipases is the preferred method for producing bioactive compounds. One strategy for obtaining highly enantioselective lipases that are stable in the organic media that are often used in these reactions is to improve existing lipases by protein engineering. In this work, we engineered the lipase LipC12, which has good stability in organic media, but only moderate enantioselectivity. Molecular docking with LipC12 identified V261 as a key position influencing, first, enantioselectivity in the transesterification of (RS)-1-phenylethanol and, second, activity in the hydrolysis of p-nitrophenyl octanoate. Variants were then obtained by site-directed mutagenesis, expressed in Escherichia coli, and their performance in these reactions was evaluated. Enzymes immobilized on Immobead 150 were used in the transesterification while free enzyme was used in the hydrolysis reaction. It was not possible to increase the hydrolytic activity and enantioselectivity simultaneously: some variants had increased enantioselectivity but lower hydrolytic activity, and others had increased hydrolytic activity but lower enantioselectivity. The best result for enantioselectivity was obtained for LipC12V261Q, with an increase of the E-value (for (R)-1-phenylethanol) from 46 to 110, however, its hydrolytic activity decreased 4-fold in comparison to LipC12wt. The highest hydrolytic activity was obtained for LipC12V261F, with a value almost 6-fold higher than that of LipC12wt. This variant also had an inverted enantiopreference (i.e. for (S)-1-phenylethanol), but with a very low E-value of only 4.

Wastewater Treatment Using Active Microorganisms and Evaluation of Results

In this study, samples are taken from the facility where the wastewater is located, and tests and analyzes are made with an active microorganism (EM). EM Technology means technology using Active Microorganisms. EM is not composed of a single microorganism, but a mixture of various groups of microorganisms. It has a positive and healthy effect on people, animals, and nature. EM is effective in both aerobic and anaerobic conditions. For this reason, it would be beneficial to apply it at all stages of treatment in conventional wastewater treatment systems, except for the biological reactor. When used in EM-activated wastewater, various compounds are formed that may be beneficial for wastewater (enzymes with high hydrolytic activity and some antioxidant substances). These compounds together with EM microorganisms provide high-capacity purification of wastewater. As a result of analyses using EM, it is seen that the desired values are achieved. Heavy metal determination is made in Çankırı wastewater and water treated with EM using an ICP-OES device at a lower detection limit (LOD) value of the device at ppm level. As a result of treatment with EM, it has been determined that a high concentration of strontium fell to the lower detection limit. This result shows that EM has an effective role in removing heavy metals. It has been seen that the use of EM in wastewater is extremely beneficial in terms of eliminating bad odors, increasing the sedimentation rate of organic matter, and reducing chemical oxygen demand (COD), biochemical oxygen demand (BOD), and suspended solids amount (SSM). Besides, ionization is suppressed by the anti-oxidation effect. When EM is used, the operating cost will be lower as O2 consumption can be reduced. Since the water will still contain EM microorganisms after being treated, it is thought that wherever it is discharged from the wastewater plant to nature, a great improvement will be observed in terms of vegetation and the environment.

Increasing of efficiency of fermentation process of organic substrates for intensification of methanogeneration

The prospects of increasing the efficiency of fermentation process of organic substrates to ensure the maximum yield of biogas and increase the economic efficiency of processing of organic sewage sludge are considered. Biogas, as an analogue of natural gas, is used in production of thermal and electrical energy, as transport motor fuel, as a raw material for chemical industry.&#x0D; An analysis of trends in intensification of methane fermentation processes was carried out. A promising way to increase productivity of methane generation process during processing of organic matter is to increase the energy saturation of an organic substrate and its preliminary treatment. One of the approaches aimed at intensifying methane generation is use of organic waste as an auxiliary substrate for fermentation of sewage sludge – a process known as co-fermentation.&#x0D; The sediments of primary and secondary settling tanks of the biological wastewater treatment complex were studied. According to results of tests, it was noted that ratio of carbon and nitrogen in the studied excess activated sludge is 4–5, in the mixture of primary and secondary sediment – 5–6, in sediment from sludge sites – 6–10, which is significantly less than desired ratio of 20:1, which limits the negative impact of increased nitrogen content in the organic substrate for methane fermentation. According to obtained data, it can be predicted that nitrogen-containing compounds present in sewage sludge will contribute to a microbiological process of formation of nitrogen-containing reduced products, which reduce output of methane during methanogenesis.&#x0D; The expediency of enriching the inoculated biocenosis with hydrolytic enzyme preparations or microorganisms with high hydrolytic activity is shown, given the fact that activated sludge has a lower hydrolytic activity than enzyme preparations isolated from the same sludge, as well as than some bacteria, and even more so microscopic fungi. This was caused by presence of inert organic and inorganic mass, dead microorganisms and those with low hydrolytic activity in the sludge biomass. It is also possible to increase speed of the first phase of sediment fermentation due to alkaline pretreatment of the substrate mixture.

Enlarging the substrate binding pocket of penicillin G acylase from Achromobacter sp. for highly efficient biosynthesis of β-lactam antibiotics

Penicillin G acylase (PGA) is a key biocatalyst for the enzymatic production of β-lactam antibiotics, which can not only catalyze the synthesis of β-lactam antibiotics but also catalyze the hydrolysis of the products to prepare semi-synthetic antibiotic intermediates. However, the high hydrolysis and low synthesis activities of natural PGAs severely hinder their industrial application. In this study, a combinatorial directed evolution strategy was employed to obtain new PGAs with outstanding performances. The best mutant βF24G/βW154G was obtained from the PGA of Achromobacter sp., which exhibited approximately a 129.62-fold and a 52.55-fold increase in specific activity and synthesis/hydrolysis ratio, respectively, compared to the wild-type AsPGA. Thereafter, this mutant was used to synthesize amoxicillin, cefadroxil, and ampicillin; all conversions > 99% were accomplished in 90–135 min with almost no secondary hydrolysis byproducts produced in the reaction. Molecular dynamics simulation and substrate pocket calculation revealed that substitution of the smallest glycine residue at βF24 and βW154 expanded the binding pocket, thereby facilitating the entry and release of substrates and products. Therefore, this novel mutant is a promising catalyst for the large-scale production of β-lactam antibiotics.

Insight of external ultrasound on energy-production acceleration from renewable Al-water reaction in Al-based metallic materials

Harnessing renewable metal fuels to supplement conventional bio-mass energy supply in certain areas is steadily demanded under the current global energy policy of CO2 emission reduction. The Al-to-power of a semi-cycle phase in the renewable conception by hydrolysis in aluminum-based metallic materials is viewed promising, yet surficial oxidation of aluminum matrices and the reaction by-products always deactivate the materials. Ultrasound induction in the hydrolysis process is proposed as an effective route to maintain a high-hydrolysis activity, but the trials on metallic materials are still blank. This article addresses the ultrasound accelerated hydrolysis of a representative aluminum-based metallic material, and the process was in-situ explored by high-speed photography to unravel the mechanism along with numerical modelling. The result shows that ultrasound fractures the particle material and exfoliates the reaction by-product layer to instantly expose more fresh aluminum, resulting in accelerated hydrogen generation. Using these discoveries, the employment of ultrasound in exploiting the hydrolysis of Al-based materials to meet the renewable metal-fuel demand is anticipated.

Three Molecular Modification Strategies to Improve the Thermostability of Xylanase XynA from Streptomyces rameus L2001

Glycoside hydrolase family 11 (GH11) xylanases are the preferred candidates for the production of functional oligosaccharides. However, the low thermostability of natural GH11 xylanases limits their industrial applications. In this study, we investigated the following three strategies to modify the thermostability of xylanase XynA from Streptomyces rameus L2001 mutation to reduce surface entropy, intramolecular disulfide bond construction, and molecular cyclization. Changes in the thermostability of XynA mutants were analyzed using molecular simulations. All mutants showed improved thermostability and catalytic efficiency compared with XynA, except for molecular cyclization. The residual activities of high-entropy amino acid-replacement mutants Q24A and K104A increased from 18.70% to more than 41.23% when kept at 65 °C for 30 min. The catalytic efficiencies of Q24A and K143A increased to 129.99 and 92.26 mL/s/mg, respectively, compared with XynA (62.97 mL/s/mg) when using beechwood xylan as the substrate. The mutant enzyme with disulfide bonds formed between Val3 and Thr30 increased the t1/260 °C by 13.33-fold and the catalytic efficiency by 1.80-fold compared with the wild-type XynA. The high thermostabilities and hydrolytic activities of XynA mutants will be useful for enzymatic production of functional xylo-oligosaccharides.

Staphylococcus aureus MazG hydrolyzes oxidized guanine nucleotides and contributes to oxidative stress resistance

We previously reported that knockout of the mazG (SA1292) gene decreases Staphylococcus aureus killing activity against silkworms. S. aureus MazG (SaMazG) has a nucleotide pyrophosphatase domain conserved among MazG family proteins, but its biochemical characteristics are unknown. In the present study, we purified recombinant N-terminal His-tagged SaMazG protein and examined its biochemical activity. SaMazG hydrolyzed GTP, UTP, dGTP, and TTP into nucleoside monophosphates. Hydrolytic activity of SaMazG against ATP, CTP, dATP, and dCTP was low or not detected. SaMazG exhibited high hydrolytic activity against 8-oxo-GTP and 8-oxo-dGTP, oxidized guanine nucleotides, with a Vmax/Km ratio more than 15-fold that of GTP. Furthermore, the S. aureus mazG knockout mutant was sensitive to hydrogen peroxide compared with the parent strain. These results suggest that SaMazG is a nucleotide pyrophosphatase hydrolyzing oxidized guanine nucleotides that contributes to the oxidative stress resistance of S. aureus.