High Hydrolytic Activity Research Articles

Relationship between pellet formation by Aspergillus oryzae strain KB and the production of β-fructofuranosidase with high transfructosylation activity

Aspergillus oryzae KB produces two β-fructofuranosidases (F1 and F2). F1 has high transfructosylation activity (Ut) to produce fructooligosaccharides. F2 has high hydrolysis activity (Uh), releasing glucose and fructose. It is desirable to selectively produce F1, which can be used for production of fructooligosaccharides. Here, the relationship between filamentous pellet size and selective production of F1 in liquid culture was investigated. Our finding revealed that: (i) The mean particle size of pellets (5.88 ± 1.36 mm) was larger, and the ratio of Ut to Uh was improved (Ut/Uh = 5.0) in 10% sucrose medium compared with 1% sucrose medium (pellet size = 2.60 ± 0.37 mm; Ut/Uh = 0.96). (ii) The final culture pH of the 1% sucrose medium was 8.7; on controlling the pH of 1% sucrose medium at 5.0, increased pellet size (9.69 ± 2.01 mm) and Ut/Uh (7.8) were observed. (iii) When 3% glycerin was used as carbon source, the pellet size decreased to 1.09 ± 0.33 mm and Ut/Uh was 0.57. (iv) In medium containing 1% sucrose, the pellet size was dependent on the number of spores used in the culture inoculum, but, in these experiments, Ut/Uh was almost constant (1.05 ± 0.08). Collectively, the data show that the value of Ut/Uh is proportional to the pellet size when liquid culture of A. oryzae strain KB is performed in some conditions (such as in the presence of high sucrose concentration, low pH, or added Tween surfactant), but in other conditions Ut/Uh is independent of pellet size.

Prediction and comparison of textural properties of magnetic copolymer supports for enzyme immobilization

AbstractMagnetic polymers supports have proven to be valuable materials for enzyme immobilization, as they allow recovering the catalyst by magnetic separation, precluding the need for costly and time‐consuming separation steps. In this study, magnetic copolymer supports were synthesized using styrene (STY) and different crosslinking agents (divinylbenzene, ethylene glycol dimethacrylate, or triethylene glycol dimethacrylate) and initiators (azobisisobutyronitrile or benzoyl peroxide) and used to immobilize Candida antarctica lipase B (CALB). The aim was to obtain biocatalysts with high enzymatic activity and satisfactory morphological properties for use in biotransformation reactions. Two morphological properties known to influence the immobilization yield were taken into consideration, specific surface area, and swelling index. Experimental data were compared to the predictions of a model based on molar balance, method of moments, numerical fractionation, and elementary gel structures. The high correlation (R2 = 0.9974) between experimental and predicted values demonstrated the suitability of the model for estimating the textural properties of enzyme supports. CALB was successfully immobilized, showing high hydrolytic activity (500–700 U g−1) and good thermal stability at 50°C. CALB/STY‐EGDMA‐M was 14 times more stable than free CALB. The results confirm the efficiency of the immobilization method and the suitability of the copolymers for enzyme immobilization.

Activity and Partial Characterization of Trypsin, Chymotrypsin, and Lipase in the Digestive Tract of Totoaba macdonaldi

ABSTRACTMolecular weights of trypsin, chymotrypsin, and lipase from anterior intestine and pyloric caeca of Totoaba macdonaldi were evaluated, as well as optimum temperature and pH for activity of the proteases. Trypsin was 24.1 kDa and effectively hydrolyzed Nα-benzoyl-DL-arginine 4-nitroanilide hydrochloride at optimum pH and temperature of 8 and 65°C, respectively. Chymotrypsin was 25.9 kDa and showed higher hydrolytic activity for N-benzoyl-L-tyrosine ethyl ester at pH 8 and 45°C, with a wider range of statistically similar activity values. Two pancreatic lipases of 70.2 and 47.5 kDa were detected, which could be the uncleaved and the final form of a colipase-dependent pancreatic lipase, since enzyme activity was detected without supplementation of bile salts and supplementing them inhibited activity.

Combined use of single molecule real-time DNA sequencing technology and culture-dependent methods to analyze the functional microorganisms in inoculated raw wheat Qu

Inoculated raw wheat Qu (IRWQ), which can be produced with high efficiency and low cost while maintaining stable quality, is a new Qu that has been applied to Huangjiu brewing. In this study, single molecule real-time DNA sequencing technology and culture-dependent methods were combined for the first time to study the microbiota and the function of the principle microorganisms in IRWQ. The glucoamylase, amylase and protease contents of IRWQ were 1.17, 1.55 and 2.87-times greater, respectively, than those of traditional wheat Qu. Like traditional wheat Qu, the main volatile flavor compounds in IRWQ were alcohols; however, the esters content was much higher and the acids content was much lower. Single molecule real-time DNA sequencing technology identified 18 fungal species and 59 bacterial species in IRWQ. Then, 30 species were isolated by culture-dependent methods. These species represented about 80% of the total fungal microbiota and 35% of the total bacteria microbiota. Molds were the main contributors of glucoamylase, amylase and protease activities. Aspergillus flavus had the highest glucoamylase and protease activity. Bacillus subtilis and Bacillus amyloliquefaciens also produced high hydrolytic enzyme activities. Saccharomyces cerevisiae produced the largest amounts of aromatics compounds, alcohols, esters and acids. Aldehydes and ketones are mainly produced by molds.

Identification and characterization of a novel phthalate-degrading hydrolase from a soil metagenomic library.

Phthalate esters have raised public concerns owing to their effects on the environment and human health. We identified a novel phthalate-degrading hydrolase, EstJ6, from a metagenomic library using function-driven screening. Phylogenetic analysis indicated that EstJ6 is a member of family IV esterases. EstJ6 hydrolyzed various dialkyl and monoalkyl phthalate esters, and exhibited high hydrolytic activity (128 U/mg) toward dibutyl phthalate at 40°C and pH 7.5. EstJ6 hydrolyzed not only common phthalate esters with simple side chains but also diethylhexyl phthalate and monoethylhexyl phthalate, which have complex and long side chains. Site-directed mutagenesis indicated that the catalytic triad residues of EstJ6 consists of Ser146, Glu240, and His270. EstJ6 is therefore a promising biodegradation enzyme, and our study illustrates the advantages of a metagenomic approach in identifying enzyme-coding genes for agricultural, food, and biotechnological applications.

A self-confinement synthesis of a POM-decorated MOF thin film for actively hydrolyzing ethyl acetate.

Hybrid heterogeneous solid acid catalysts polyoxometalate-decorated metal organic framework (POM@MOFs) thin films were synthesized from POMs and metal hydroxide nanostrands via a self-confinement method at room temperature, which show high hydrolyzing activity (1027.5 μmol g-1 min-1) for ethyl acetate with durable stability and an easy recovery. This provides a general way to easily prepare nano POM@MOF thin film catalysts.

A novel lipase from Aspergillus oryzae catalyzed resolution of (R,S)-ethyl 2-bromoisovalerate.

In this study, a novel lipase M5 derived from Aspergillus oryzae WZ007 was prone to exhibit high hydrolytic activity and stereoselectivity towards racemic substrate (R,S)-ethyl 2-bromoisovalerate. (R)-ethyl 2-bromoisovalerate was obtained by enzymatic resolution, which is the key chiral intermediate for highly efficient enantiomerically fluvalinate. The results showed that the enzymatic reaction was carried out in 120mM racemic substrate for 3 hours, the enantiomeric excess reached 98.6%, the conversion was 51.7%, and E value above 120. Therefore, the novel lipase M5 has the ability to efficiently produce (R)-ethyl 2-bromoisovalerate, which greatly reduces the industrial production cost of the highly efficient counterpart of fluvalinate.

High intrinsic hydrolytic activity of cyanobacterial RNA polymerase compensates for the absence of transcription proofreading factors.

The vast majority of organisms possess transcription elongation factors, the functionally similar bacterial Gre and eukaryotic/archaeal TFIIS/TFS. Their main cellular functions are to proofread errors of transcription and to restart elongation via stimulation of RNA hydrolysis by the active centre of RNA polymerase (RNAP). However, a number of taxons lack these factors, including one of the largest and most ubiquitous groups of bacteria, cyanobacteria. Using cyanobacterial RNAP as a model, we investigated alternative mechanisms for maintaining a high fidelity of transcription and for RNAP arrest prevention. We found that this RNAP has very high intrinsic proofreading activity, resulting in nearly as low a level of in vivo mistakes in RNA as Escherichia coli. Features of the cyanobacterial RNAP hydrolysis are reminiscent of the Gre-assisted reaction—the energetic barrier is similarly low, and the reaction involves water activation by a general base. This RNAP is resistant to ubiquitous and most regulatory pausing signals, decreasing the probability to go off-pathway and thus fall into arrest. We suggest that cyanobacterial RNAP has a specific Trigger Loop domain conformation, and isomerises easier into a hydrolytically proficient state, possibly aided by the RNA 3′-end. Cyanobacteria likely passed these features of transcription to their evolutionary descendants, chloroplasts.

Efficient synthesis of L-phosphinothricin using a novel aminoacylase mined from Stenotrophomonas maltophilia

L-phosphinothricin (L-PPT) is a competitive and environmentally friendly herbicide. To develop an efficient approach for synthesis of l-PPT, a kinetic resolution route with a novel aminoacylase (SmAcy) mined from Stenotrophomonas maltophilia using N-acetyl-PPT as a substrate was first constructed. This SmAcy exhibited high hydrolytic activity and excellent enantioselectivity (E > 200) toward N-acetyl-PPT. Optically pure l-PPT (> 99.9 % eep) was acquired with high conversion (> 49 %) within 4 h by the whole cells. On the basis of the docking analysis, a main reason for high enantioselectivity (E > 200) of SmAcy towards l-enantiomer would be that the D-N-acetyl-PPT cannot interact with the key general acid-base residue and the metal ions. A low-cost and simple preparation process of the substrate from commercially available racemic PPT for production of L-PPT was provided. A chemical racemization method of the unreacted D-enantiomer of substrate was also provided to recycle the unwanted substrate enantiomer. This study provides a potential route for the industrial production of L-PPT.

Hydrolysis extraction of diosgenin from Dioscorea nipponica Makino by sulfonated magnetic solid composites

Extraction of diosgenin from the rhizome of Dioscorea by catalytic hydrolysis over recyclable solid acids is one of the most enviromentally friendly ways for the conversion of biomass into chemicals. In this paper, a magnetic solid acid, Fe3O4@SiO2@NH-(CH2)2-NH2@SO3H with the particle size of about 80 nm, was synthesized by using Fe3O4 (~ 20 nm) as a magnetic core and then coated orderly with tetraethyl orthosilicate (TEOS) and N-{3-(Trimethoxysilyl) propyl} ethylenediamine (TMPED) by sol-gel reactions in water/ethanol solution, followed by the sulfonation of chlorosulfonic acid. The solid acid was characterized by Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), scanning electron microscope (SEM), X-ray diffraction (XRD), vibrating sample magnetometer (VSM), and X-ray photoelectron spectroscopy (XPS). The content of acidic sites on the surface of the solid acid was 1.01 mmol/g, measured by back titration method. The prepared solid acid was used to hydrolyze and extract diosgenin from Dioscorea nipponica Makino (DNM). The results show that the magnetic solid acid has higher hydrolysis activity than 2.5 M hydrochloric acid under same hydrolysis conditions, at 110 °C for 5 h. In addition, the magnetic solid acid can be easily separated from the reaction mixture by the application of a magnet and reused several times without significant activity loss. This work has a potential application value for the extraction of diosgenin from plants.

Kinetic study of NaBH4 catalytic hydrolysis using supported NiCo2O4

Supported NiCo2O4 catalyst (NiCo2O4/zeolite) was prepared by homogeneous co-precipitation combined with a sintering method. The NiCo2O4/zeolite catalyst was characterized by XRD, BET, FESEM coupled with EDS, and XPS. The results indicated that NiCo2O4 microspheres produced excellent growth on the zeolite, constructing porous structures. Moreover, the NiCo2O4/zeolite catalyst exhibited high activity for NaBH4 hydrolysis (experiment showed that the zeolite carrier had no catalytic activity for NaBH4 hydrolysis); the hydrogen generation rate was as high as ∼6219 ml·min-1·g-1. Furthermore, the kinetics of NaBH4 hydrolysis was systematically studied by changing the temperature, the NiCo2O4/zeolite catalyst dosage, concentrations of NaOH and NaBH4. According to the power law model, the overall kinetic rate expression of NaBH4 hydrolysis using NiCo2O4/zeolite catalyst was Additionally, the NiCo2O4/zeolite catalyst was highly recyclable which demonstrated its durability , making this material suitable for practical applications in PEMFC and portable devices.

Dual-functional acyl chloride monomer for interfacial polymerization: Toward enhanced water softening and antifouling performance

Here, a strategy to control surface negative charge, pore size and active layer thickness by manipulating the polyamide (PA) layer structure is proposed to fabricate an advanced water softening nanofiltration (NF) membrane. Based on this strategy, a dual-functional acyl dichloride monomer containing a protected amino group, 5-sulfinyl amino isophthaloyl dichloride (NSO), is designed as organic phase alternative. Because the protected amine group of NSO has higher hydrolytic activity and is prone to de-protect and form free amino group, it has important impact on the interfacial polymerization (IP) process and formed polyamide (PA) structure. To elucidate the role of the protected amino group in regulating PA structure, the desalination performances and structural characteristics of the three PA membranes prepared by NSO monomer and its structurally similar monomers (isophthalyl chloride (IPC) and trimesoyl chloride (TMC)) are systematically investigated. Compared with TMC-based NF membrane, the NSO-based membrane shows a smoother and lower carboxyl group density surface, decreased pore size and a thinner PA rejection layer (~30 nm). As a result, the novel NF membrane exhibits outstanding water softening performance in terms of ultrahigh divalent salts (Na2SO4, MgSO4, MgCl2 and CaCl2) rejections (above 98%) and preferable high water flux (138.25 ± 6.32 LMH), together with superior antifouling property. More importantly, its water softening capability remain stable even under the mixed salt solution including abundant SO42−, which far outperforms the typical poly(piperazine-amide)-based and state-of-the-art commercial NF membranes. This work further establishes the structure-property relationship and enables the rational design of high performance of NF membrane at the molecular level.

Molecular characterization and functional analysis of a novel candidate of cuticle carboxylesterase in Spodoptera exigua degradating sex pheromones and plant volatile esters

Odorant-degrading enzymes (ODEs) are considered to play key roles in odorant inactivation to maintain the odorant receptor sensitivity of insects. Some members of carboxylesterase (CXE) is a major sub-family of ODEs. However, only a few CXEs have been functionally characterized so far. In the present study, we cloned the antennal esterase SexiCXE11 cDNA full-length sequences from the male antennae of a notorious crop pest, Spodoptera exigua, and its encoded 538 amino acids. It was similar to other insect esterases and had the characteristics of a carboxylesterase. We expressed recombinant enzyme in High-Five insect cells and obtained the high level purified recombinant protein by affinity column. Furthermore we test enzyme activity toward its two acetate sex pheromone components (Z9,E12-Tetradecadienyl acetate, Z9E12-14:Ac and Z9-Tetradecenyl acetate, Z9-14:Ac) and other 18 ester plant volatiles. Our results demonstrated that SexiCXE11 degraded acetate sex pheromone components with similar degradation activities (about 15.75% with Z9E12-14:Ac and 19.28% with Z9-14:Ac) and plant volatiles with a relatively high activity such as pentyl acetate and (Z)-3-hexenyl caproate. SexiCXE11 had high hydrolytic activity with these two ester odorants (>50% degradation), which is characterized that although a ubiquitous expression esterase SexiCXE11 may be partly involved with olfaction. This study may facilitate a better understanding of moth ODE differentiation and suggest strategies for the development of new pest behavior inhibitors.

Agroindustrial waste as a resource for volatile fatty acids production via anaerobic fermentation

This study evaluated the feasibility of the anaerobic digestion as a sustainable valorisation strategy for volatile fatty acids production from agroindustrial waste (cucumber, tomato and lettuce). High bioconversion efficiencies were reached by operating the reactors at 25 °C, 3 g VS·d−1·L−1 with pH adjustment. Cucumber fermentation achieved the highest bioconversion (52.6%), whereas tomato degradation was the least efficient bioprocess (40.1%) due to the low pH (5.6) that partially inhibited the hydrolytic and acidogenic activities. In all cases, carboxylic acid profiles were mainly composed of volatile fatty acids with even carbon number. The developed microbial community exhibited high hydrolytic and acidogenic activities associated to carbohydrates degradation. This microbial population was dominated by Firmicutes phylum and showed a lack of acetogenic bacteria related with CH4 production, resulting in a remarkably high VFAs accumulation.

Extraction and immobilization of Proteolytic Enzyme from local Yemeni Bean Seeds (Dolichos Lablab L.)

The aim of this study is to prepare immobilized protease with high hydrolytic activity forbiotechnological applications. Local Bean Seeds (Dolichos Lablab L.) were used for the extraction of acidprotease using tris-buffer, pH 4.5 as enzyme solvent. Free acidic protease was immobilized on entrapment in calcium alginate gel (in situ activated) by covalent binding method. Their activity and immobilization efficiency for hemoglobin hydrolysis was investigated. Temperature and pH maxima of the immobilized protease showed no changes before and after immobilization. The immobilized protease exhibited good thermal stability and re-usability.

A Novel VIII Carboxylesterase with High Hydrolytic Activity Against Ampicillin from a Soil Metagenomic Library.

A novel carboxylesterase gene, named dlfae4, was discovered and sequenced from a soil metagenomic library. The dlfae4 gene was composed of 1017 base pairs encoding 338 amino acid residues with a predicted molecular mass of 37.2kDa. DLFae4 exhibited strong hydrolytic activity towards methyl ferulate under optimum pH and temperature conditions (pH 8.6, 50°C) and displayed remarkable thermostability, with residual activity as high as 50% after incubation for 3h at 60°C. A family VIII esterase DLFae4 was found to contain a typical serine residue within the S-X-X-K motif, which serves as a catalytic nucleophile in class C β-lactamases and family VIII esterases. As a consequence of its high sequence similarity with β-lactamases, DLFae4 exhibited significant hydrolytic activity towards ampicillin. In addition, DLFae4 was found to be the first known member of family VIII carboxylesterases with phthalate-degrading ability. Site-directed mutagenesis studies revealed that Ser11, Lys14, and Tyr121 residues play an essential catalytic role in DLFae4. These new findings, which are of great importance for further in-depth research and engineering development of carboxylesterases, should advance the implementation of biotechnological applications.

Unravelling the diversity of glycoside hydrolase family 13 \u03b1-amylases from Lactobacillus plantarum WCFS1

Backgroundα-Amylases specifically catalyse the hydrolysis of the internal α-1, 4-glucosidic linkages of starch. Glycoside hydrolase (GH) family 13 is the main α-amylase family in the carbohydrate-active database. Lactobacillus plantarum WCFS1 possesses eleven proteins included in GH13 family. Among these, proteins annotated as maltose-forming α-amylase (Lp_0179) and maltogenic α-amylase (Lp_2757) were included.ResultsIn this study, Lp_0179 and Lp_2757 L. plantarum α-amylases were structurally and biochemically characterized. Lp_2757 displayed structural features typical of GH13_20 subfamily which were absent in Lp_0179. Genes encoding Lp_0179 (Amy2) and Lp_2757 were cloned and overexpressed in Escherichia coli BL21(DE3). Purified proteins showed high hydrolytic activity on pNP-α-D-maltopyranoside, being the catalytic efficiency of Lp_0179 remarkably higher. In relation to the hydrolysis of starch-related carbohydrates, Lp_0179 only hydrolysed maltopentaose and dextrin, demonstrating that is an exotype glucan hydrolase. However, Lp_2757 was also able to hydrolyze cyclodextrins and other non-cyclic oligo- and polysaccharides, revealing a great preference towards α-1,4-linkages typical of maltogenic amylases.ConclusionsThe substrate range as well as the biochemical properties exhibited by Lp_2757 maltogenic α-amylase suggest that this enzyme could be a very promising enzyme for the hydrolysis of α-1,4 glycosidic linkages present in a broad number of starch-carbohydrates, as well as for the investigation of an hypothetical transglucosylation activity under appropriate reaction conditions.

Bridges to Stability: Engineering Disulfide Bonds Towards Enhanced Lipase Biodiesel Synthesis

AbstractComputational design of disulfide bonds was performed for lipase from Geobacillus stearothermophilus T6 (LipT6) for enhanced methanol stability and improved biodiesel production. Thirteen double mutants comprising new cysteine pairs were screened and evaluated for their stability in 70 % methanol. Superior stability was found with variant E251C/G332C (M13) having a 5.5‐fold higher hydrolysis activity and enhanced unfolding temperature (Tm) of +7.9 °C in methanol compared with wild‐type. Moreover, M13 converted nearly 80 % waste chicken oil to biodiesel, representing a 2.4‐fold improvement relative to the WT. Structural studies using X‐ray crystallography confirmed the existence of the engineered disulfide bonds shedding light on the link between the bond location and backbone architecture with its stabilization impact. Rational integration of disulfide bonds is suggested to be a feasible method to promote elevated stability in organic solvents for various industrial applications such as biodiesel synthesis.

A new β-galactosidase extracted from the infant feces with high hydrolytic and transgalactosylation activity.

A β-galactosidase (β-GalINF) was directly isolated from feces of an 8-month-old infant. Mass spectrum analysis showed β-GalINF with coverage over 50% to the β-galactosidase from Bifidobacterium longum EK3. Accordingly, the β-galINF was amplified from the feces metagenomic DNA by degenerate primers. After overexpressed in Escherichia coli, the β-GalINF was purified and biochemical characterized. β-GalINF existed as homotetramer and homodimer, whose activity (optimal at 50 °C, pH 6.5) was exhilaratingly increased to 484% by artificial intestinal juice. The Km and Vmax values for oNPG and lactose were 20.95 ± 2.76 mM, 5004.50 ± 318.8 μmol min-1 mg-1 and 140.2 ± 17.7 mM, 293.1 ± 14.7 μmol min-1 mg-1, respectively. The production rate of galacto-oligosaccharides by β-GalINF from 20% lactose at 50 °C was 33.4 ± 0.67%. These results suggested the β-GalINF with high hydrolytic and transgalactosylation activity from the infant intestinal has great potential as infant lactase preparation. Moreover, this study provided a new way for exploring undetected enzymes by uncultured-dependent methods.

Active Site Architecture and Reaction Mechanism Determination of Cold Adapted β-d-galactosidase from Arthrobacter sp. 32cB.

ArthβDG is a dimeric, cold-adapted β-d-galactosidase that exhibits high hydrolytic and transglycosylation activity. A series of crystal structures of its wild form, as well as its ArthβDG_E441Q mutein complexes with ligands were obtained in order to describe the mode of its action. The ArthβDG_E441Q mutein is an inactive form of the enzyme designed to enable observation of enzyme interaction with its substrate. The resulting three-dimensional structures of complexes: ArthβDG_E441Q/LACs and ArthβDG/IPTG (ligand bound in shallow mode) and structures of complexes ArthβDG_E441Q/LACd, ArthβDG/ONPG (ligands bound in deep mode), and galactose ArthβDG/GAL and their analysis enabled structural characterization of the hydrolysis reaction mechanism. Furthermore, comparative analysis with mesophilic analogs revealed the most striking differences in catalysis mechanisms. The key role in substrate transfer from shallow to deep binding mode involves rotation of the F581 side chain. It is worth noting that the 10-aa loop restricting access to the active site in mesophilic GH2 βDGs, in ArthβDG is moved outward. This facilitates access of substrate to active site. Such a permanent exposure of the entrance to the active site may be a key factor for improved turnover rate of the cold adapted enzyme and thus a structural feature related to its cold adaptation.