Hydrolytic Ability Research Articles

Effect of inoculum composition on the microbial community involved in the anaerobic digestion of sugarcane bagasse

ABSTRACT In anaerobic digestion (AD), the choice of inoculum type seems to be relevant for methane production for complex substrates, such as lignocellulosic material. Previous work demonstrated that the addition of fresh manure and ruminal fluid to anaerobic sludge improved methane productivity and kinetics of AD of crude sugarcane bagasse (CSB). Considering that the improvement of methane production could be a result of a more adapted microbial community, the present study performed the Next Generation Sequencing analysis to identify changes in the microbiome of anaerobic sludge inoculum, resulting from fresh manure and ruminal fluid addition. In comparison with AD performed only with anaerobic sludge inoculum (50:50, U), accumulated methane production was 15% higher with anaerobic sludge plus ruminal fluid inoculum (50:50, UR) and even higher (68%) with anaerobic sludge with fresh bovine manure inoculum (50:50, UFM), reaching the value of 143 NmLCH4.gVS −1. Clostridium species were highly abundant in all inocula, playing an important role during the hydrolysis and fermentation of CSB, and detoxifying potential inhibitors. Microbial composition also revealed the occurrence of Pseudomonas and Anaerobaculum at UFM inoculum that seem to have contributed to the higher methane production rate, mainly due to their hydrolytic and fermentative ability on lignocellulosic substrates. On the other hand, the presence of Alcaligenes might have had a negative effect on methane production due to their ability to perform methane oxidation.

Improved sensitivity of gold nanoclusters toward trypsin under synergistic adsorption of CdTe quantum dots

Many physiological and pathological processes are associated with trypsin, so the sensitive detection of trypsin is significant for exploring these processes. Herein, AuNCs/CdTe QDs as the complex of bovine serum albumin-capped gold nanoclusters (BSA-AuNCs) with 3-mercaptopropionic acid-stabilized CdTe quantum dots (MPA-CdTe QDs) were prepared via electrostatic assembly for sensitive determination of trypsin through synergistic effect. In AuNCs/CdTe QDs, the red luminescence of BSA-AuNCs kept almost unchanged, whereas the green luminescence of MPA-CdTe QDs disappeared because of the static quenching process. Once trypsin was added to AuNCs/CdTe QDs, the structure of BSA-AuNCs was frustrated ascribed to the hydrolysis ability of trypsin toward BSA, causing the red luminescence of BSA-AuNCs to quench, moreover the electronic attraction of MPA-CdTe QDs toward trypsin facilitated the hydrolysis of BSA. The detection sensitivity of AuNCs/CdTe QDs for trypsin was therefore promoted under the synergistic adsorption of MPA-CdTe QDs, and the detection limit of trypsin with AuNCs/CdTe QDs was determined as low as 12 ng mL−1 (0.50 nM). AuNCs/CdTe QDs were employed to the measurement of trypsin in urine samples, and the result indicated that spiked recoveries ranging from 80.0 % to 119 % were acquired, offering a reliable tool to explore the relevant physiological and pathological processes.

Evaluation of Non-Conventional Yeasts Isolated from Rotten Wood for Hydrolytic Activities and Xylose Fermentation

Hydrolysis of lignocellulose to fermentable sugars and their subsequent conversion to ethanol remain great challenges in the biofuel industry. Rotten wood is first colonized by bacteria and molds that possess strong hydrolases. Yeasts are also an important group of microorganisms that may participate in wood hydrolysis. Decaying wood could provide a rich natural reservoir of yeasts possessing promising hydrolytic activities, including xylanases, cellulases, β-glucosidases, or abilities essential for the fermentation of pentose sugars derived from lignocellulose degradation, especially xylose. Therefore, the aim of this work was to screen yeasts isolated from rotten wood samples for the production of hydrolytic enzymes directed at lignocellulose components and the ability to ferment xylose, L-arabinose, and cellobiose. Methods. Yeast strains were isolated from 22 samples of rotten wood and identified by phenotypic characteristics according to Kurtzman et al. Hydrolytic properties and the ability of the isolated strains to ferment xylose, L-arabinose, and cellobiose were determined using conventional methods. Results. 30 strains of yeasts and yeast-like micromycetes were isolated from 22 samples of rotten wood in the Holosiivskyi Forest, Kyiv. Based on phenotypic properties, most of the isolated yeasts belonged to ascomycetous yeasts and were represented by the following genera: Candida (8 strains), Debaryomyces (5 strains), Kluyveromyces (5 strains), Pichia (5 strains), Scheffersomyces (2 strains), Lachancea, Hanseniaspora, Saccharomyces, and Geotrichum/Galactomyces. A strain of yeast-like non-photosynthetic alga Prototheca sp. was also detected. Most of the isolated microfungi (66.6% isolates) exhibited extracellular β-glucosidase activity, two Candida tropicalis strains possessed weak pectinase and xylanase activity. None of the isolates demonstrated extracellular cellulase activity. Two yeast strains preliminarily identified as Scheffersomyces stipitis were able to ferment xylose at a concentration of 20—100 g/L over a wide temperature range up to 37°C. Acetic acid at 0.25—1% (v/v) concentration resulted in the complete inhibition of xylose fermentation. Ethanol production from xylose up to 6 g/L was observed under the microaerobic fermentation conditions for 24 hr at the substrate concentration 40 g/L, but the subsequent fermentation resulted in decreasing ethanol concentration presumably due to ethanol re-assimilation. None of the isolated strains was capable of fermenting cellobiose or L-arabinose under the microaerobic conditions. Conclusions. This work provides the characterization of yeast microbiota of rotten wood that was represented predominantly by ascomycetous yeasts. The dominant extracellular hydrolytic activity of the isolates was β-glucosidase. This is the first report on the isolation of xylose-fermenting yeasts Scheffersomyces stipitis in Ukraine, which comprised 7% of all the microfungi isolated from rotten wood.

High Molecular Weight α-Galactosidase from the Novel Strain Aspergillus sp. D-23 and Its Hydrolysis Performance

Aspergillus sp. D-23 was obtained by ultraviolet-diethyl sulfate (UV-DES) compound mutagenesis from Aspergillus sp. C18 that the α-galactosidase was purified from. According to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and non-denaturing polyacrylamide gel electrophoresis (Native-PAGE), the purified enzyme demonstrated apparent homogeneity. The monomeric α-galactosidase’s native molecular weight was 125 kDa. The optimal temperature of α-galactosidase was 65 °C, and 75% of the initial enzyme activity could be maintained between 45 and 55 °C. Its optimal pH was 5.0 with good pH stability. After incubating for 2 h at pH 3.0–8.0, it could retain more than 80% of its original activity. Different concentrations of metal ions had different effects on the α-galactosidase activity. High concentrations of Cu2+ could strongly inhibit enzyme activity and low concentrations of Fe2+ could promote enzyme activity. Additionally, as shown by thin layer chromatography and high-performance liquid chromatography, the enzyme also had good hydrolysis ability, which could efficiently hydrolyze melibiose and raffinose by more than 95%. Therefore, these excellent characteristics could make α-galactosidase a good candidate for the food and feed industries.

Expression and characterization of a bifunctional thermal β-glucosidase IuBgl3 from thermophilic archaeon Infirmifilum uzonense

β-glucosidase has important applications in food, medicine, biomass conversion and other fields. Therefore, exploring β-glucosidase with strong stability and excellent properties is a research hotspot. In this study, a GH3 family β-glucosidase gene named Iubgl3 was successfully cloned from Infirmifilum uzonense. Sequence analysis showed that the full length of Iubgl3 was 2 106 bp, encoding 702 amino acids, with a theoretical molecular weight of 77.0 kDa. The gene was cloned and expressed in E. coli and the enzymatic properties of purified IuBgl3 were studied. The results showed that the optimal pH and temperature for pNPG hydrolysis were 5.0 and 85 ℃, respectively. The enzyme has good thermal stability, and more than 85% of enzyme activity can be retained after being treated at 80 ℃ for2 h. This enzyme has good pH stability and more than 85% of its activity can be retained after being treated at pH 4.0-11.0 for 1 h. It was found that the enzyme had high hydrolysis ability to p-nitrophenyl β-d-glucoside (pNPG) and p-nitrophenyl β-d-xylopyranoside (pNPX). When pNPG was used as the substrate, the kinetic parameters Km and Vmax were 0.38 mmol and 248.55 μmol/(mg·min), respectively, and the catalytic efficiency kcat/Km was 6 149.20 s-1mmol-1. Most metal ions had no significant effect on the enzyme activity of IuBgl3. SDS completely inactivated the enzyme, while EDTA increased the enzyme activity by 30%. This study expanded the β-glucosidase gene diversity of the thermophilic archaea GH3 family and obtained a thermostable acid bifunctional enzyme with good industrial application potential.

Selection of prospective probiotic bacteria from the intestines of Rabbitfish, Siganus guttatus as a fermenter for fish feed raw materials

Rabbitfish is classified as herbivorous fish by utilizing plants as a staple food, so it has the potential to contain microflora in the digestive tract. The study aimed to select bacteria from the intestines of Rabbitfish as probiotic candidates for degradation of feed ingredients. At the beginning of the study, microbial dissolution of the intestines of 10 Rabbitfish measured weight and body length 127 ±20.1 g and 191.4±1.17 cm, healthy and not deformed, obtained from the waters around the island of Salemo, Regency. Pangkep. At this stage, they have successfully isolated 72 isolates. Then make a selection of bacteria based on the activity of extracellular enzymes by testing the hydrolysis ability of starch using Zobel media adding 1% soluble starch, protein hydrolysis using Zobel media adding 1% skim milk, cellulose hydrolysis using Zobel media adding 1% CMC. The ability of the insulator to hydrolyze starch ranges from 0.4 cm; to protein to 2.4 cm; cellulose to 1.5 cm. Based on these results, 10 isolates were selected as probiotic candidates, namely 411; 413; 422; 427; 430; 434; 437; 452 and 455. The next stage of the pathogenicity test of 10 selected isolates. Each isolate with a density of 106 CFU/mL was injected into 5 juveniles of healthy Rabbitfish intramuscularly as much as 0.1 mL. Monitoring is carried out daily for 10 days after infection to evaluate clinical signs of the disease in test fish. The results obtained showed that no symptoms of pain, injury, or death were found, so it was concluded that the bacterial isolate was not pathogenic.

Construction of Z-scheme CoAl-LDH/Bi2MoO6 heterojunction for enhanced photocatalytic degradation of antibiotics in natural water bodies

Heterojunction construction is considered to be an effective method for improving photocatalytic efficiency. Herein, Z-scheme heterojunction CoAl-LDH/Bi2MoO6 was synthesized by a simple hydrothermal method. Due to the strong hydrolysis ability of Bi3+ made the solution acidic, to prevent the solution of CoAl-LDH, the reaction solution was adjusted to neutral by NaOH solution. The prepared materials were characterized by a series of analytical techniques and their photocatalytic performance was evaluated by photocatalytic degradation of antibiotics under natural aqueous conditions (seawater and the water from the Xiangjiang River). The composite with 10% CoAl-LDH (CBA-10) displayed the optimal degradation efficiency and still had good photocatalytic activity in seawater and Xiangjiang water compared to the pure water for degradation of antibiotics. According to the photoluminescence (PL) and electrochemical impedance spectroscopy (EIS) test results, the heterojunction formed between CoAl-LDH and Bi2MoO6 effectively promoted the separation and migration of electrons and holes, thus increasing the photocatalytic degradation efficiency. In addition, the degradation efficiency of the composite with 10% CoAl-LDH under pure water conditions was 85.98% and 72.69% for tetracycline and ciprofloxacin respectively after five cycles of testing, which showed that the composite with 10% CoAl-LDH had good stability and reusability. This work would contribute to the development of composite materials for photocatalytic degradation of contaminants in natural water bodies.

Smartphone-based colorimetric sensor array using gold nanoparticles for rapid distinguishment of multiple pesticides in real samples

A simple, sensitive method for pesticide distinguishment based on a colorimetric sensor array using diverse gold nanoparticles (AuNPs) at room temperature is presented in this study. Acetylcholinesterase (AChE) hydrolysis ability was influenced by different pesticides and produced different concentrations of thiocholine by hydrolyzing acetylthiocholine iodide (ATCh). Thiocholine could be easily linked to the AuNPs through an Au − S covalent bond, and AuNPs underwent aggregation, resulting in a visible color change due to alteration of surface plasmon resonance properties. Based on these results, we successfully distinguished eight pesticides (glyphosate, thiram, imidacloprid, tribenuron methyl, nicosulfuron, thifensulfuron methyl, dichlorprop, and fenoprop) utilizing five different AuNPs by colorimetric assay. The limit of detection (LOD) of this visual method for all pesticides was less than 1.5 × 10-7 M, which was more sensitive than the U.S. Environmental Protection Agency regulations specify (1.18 ∼ 3.91 × 10-6 M). This method was further improved by combining a portable smartphone device with a color picking application using (color name AR) and RGB (red, green, blue) values. The method was successfully applied to pesticide residue distinguishment in real samples by linear discriminant analysis (LDA).

CeO2@NC nanozyme with robust dephosphorylation ability of phosphotriester: A simple colorimetric assay for rapid and selective detection of paraoxon

Enzyme-mediated dephosphorylation reaction is the important approach to realize the inactivation and detection of hazardous phosphate chemicals. To date, many phosphatases-like nanozymes (e.g., CeO2) have demonstrated the catalytic hydrolysis ability of the phosphomonoesters, rather than phosphotriester, and the CeO2 nanozyme only work under relatively harsh conditions of high temperature, and large dosage. Thus, exploration of efficient nanozymes for the rapid dephosphorylation of phosphotriester under mild conditions remains a challenge. Here, a novel CeO2@NC nanozyme is developed with excellent phosphatases-like activity based on substrate synergistic effect, in which, CeO2 nanoparticles embedded in N-doped carbon (NC) material. Taking paraoxon as the model substrate, such CeO2@NC nanozyme can drive rapid dephosphorylation of phosphotriester over a broad temperature range, which not only significantly outperforms natural phosphatases and neat CeO2, but also can preserve >80% of the optimal activity after exposure of harsh conditions, such as strong acidic/basic medium, high temperature of up to 80 °C. The excellent catalytic performance could be due to that Ce(IV)/Ce(III) species act as the active sites to realize the polarization and hydrolysis of P–O bond while NC template works as the synergistic group to adsorb the substrate. Furthermore, a simple colorimetric assay is developed for the rapid and selective detection of paraoxon. Overall, this work not only develops a highly efficient phosphatases-like nanozyme via substrate synergetic strategy, but also opens an interesting avenue for the rapid detection of organophosphorus pesticides.

Gelatins and antioxidant peptides from Skipjack tuna (Katsuwonus pelamis) skins: Purification, characterization, and cytoprotection on ultraviolet-A injured human skin fibroblasts

For full use of Skipjack tuna (Katsuwonus pelamis) canning by-products, gelatins (STG) were extracted from tuna skins using acid (STG-A), enzyme (STG-E) and hot water (STG-H) methods with yields of 65.30 ± 1.56%, 39.83 ± 1.61%, and 50.97 ± 1.44%, respectively. Due to the high imino acid contents (228, 216, and 213 residues/1000 residues for STG-A, STG-E, and STG-H, respectively), STG-A showed the highest transparency and gel strength properties. Amino acid analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), ultraviolet (UV) and Fourier transform infrared spectroscopy (FTIR) analysis confirmed that STG-A, STG-E, and STG-H kept the main structure of type I collagen, but enzyme and hot water extraction methods showed much stronger hydrolysis ability on α and β chains. Moreover, neutrase hydrolysate (STG-AH) of STG-A showed the highest 1,1-diphenyl-2-picrylhydrazyl radical (DPPH•) scavenging activity and the fraction STG-AH-І (<3.5 kDa) from STG-AH prepared by ultrafiltration could significantly protect human skin fibroblasts (HSFBs) against ultraviolet-A (UVA) injury. Furthermore, nineteen peptides with high antioxidant activity were purified from STG-AH-І and identified. These results suggested that STG-AH and its derived antioxidant peptides could serve as potential ingredients applied in health benefiting products for preventing UVA injury.

ASSESSEMENT OF PURIFIED COLLAGENASE INHIBITION ACTIVITY FROM STAPHYLLOCOCCUS AUREUS HN77 BY SOME LOCAL PLANTS EXTRACT

This study was aimed to inhibition of purified collagenase produced from S. aureus by local plants inhibitors (fruits and leaves). Sixty two local isolates of S. aureus which were isolated and identified by biochemical test subjected to the primary and secondary screening processes to select the active S. aureus isolate for collagenase production. Among these isolates, forty nine isolates with maximum zone of hydrolysis ability in primary screening (the ratio of Z/G more than 10 mm) were selected for secondary screening. It has been found that S. aureus HN77 had the highest productivity of the enzyme (49.122 U/mg protein). The selected isolate with highest level of collagenase activity was identified as S. aureus HN77 according to vitek test. The optimum conditions of collagenase production by selected isolate using submerged fermentation were obtained with medium (D) contain collagen as the best production medium, 2% starch as the best carbon source, 2% yeast extract/peptone as nitrogen source, temperature 37 °C and pH 7, after 48 hr. of incubation period, the specific activity was reached to1502.15 U/mg. The enzyme was purified by gel filtration chromatography using Sephadex G-150 then concentration by sucrose. The results shown an increase in final purification folds 6.1 time with an enzyme yield of 85.2%.The purified enzyme was exhibited maximal activity and stability at pH 7.0. The optimal temperature for purified enzyme activity was 45°C and it was stable until 45°C.The highest rate of enzyme specificity found with collagen. Collagenase was inhibited with local plants extract (fruit and leaves). Fruit of black fig extract inhibit 90% of collagenase activity while the inhibition efficiency of collagenase with grape leaf was reached to 12%.

Kecambah: Agen penghidrolisis pati yang potensial

Starch is a group of complex carbohydrates containing amylose and amylopectin. Starch can be broken down into simpler molecules such as maltose, isomaltose, and glucose which is called the hydrolysis process. Starch hydrolysis is very useful in several food industries. Enzymatic hydrolysis of starch usually uses the amylase enzyme derived from saliva. However, if you are going to carry out the hydrolysis process on a large scale, it requires large amounts of enzymes. Therefore, this study aimed to examine the ability of enzymes in sprouts to hydrolyze starch and compare it with the ability of starch hydrolysis by saliva and HCl. The isolated sprout samples were added to the starch solution and then incubated at 37oC. Every 5 minutes the mixture was tested qualitatively with Benedict's test and iodine. The same treatment for saliva and HCL. However, HCl was incubated at 100oC. The results of the qualitative test using Benedict's test and the iodine test showed enzymatic process had a higher ability to hydrolyze starch than chemically with HCl. The α-amylase enzyme produced from sprouts showed a higher starch hydrolysis ability than saliva. Sprouts are a natural source that can be developed as a starch hydrolyzing agent.

Production of cellulase by Aspergillus niger through fermentation of spent mushroom substance: Glucose inhibition and elimination approaches

In this study, the best activity of Aspergillus niger cellulase produced from spent mushroom substance (SMS) was 18.82 U/mL, and its enzymatic hydrolysis ability was similar to that of commercial cellulase (120.70 U/g) at the same enzyme loading. Moreover, A. niger cellulase remained stable at pH and temperature ranges of 4.0–8.0 and 40–60 ℃, respectively. Subsequently, the feedback inhibition by glucose on A. niger cellulase was analysed. When the glucose concentration reached 30 g/L, the activation energy of denaturation was 10.69% lower than that of the group with 0 g/L glucose, indicating that 30 g/L of glucose worsened the stability and decreased the activity of the enzyme. However, the kinetic results showed that glucose inhibition of A. niger cellulase was reversible. A. niger cellulase was used in the lactic acid (LA) fermentation of SMS. The activity of A. niger cellulase inhibited by sugars was recovered through sugar consumption by lactic acid bacteria in separate hydrolysis and fermentation, and the final saccharification yield increased from 81.04% to 87.71%. Under simultaneous saccharification and fermentation, the inhibition was avoided because the sugar concentration was < 10 g/L. Therefore, the final LA concentration and productivity were 31.98 g/L and 0.89 g/L/h, respectively.

Evolution of conformation and thermal properties of bovine hides collagen in the sodium sulphide solution

Conformations of secondary and super secondary structure from bovine hides collagen are very important for constructing collagen molecular space structure, and directly related to its properties. In this work, the evolutional mechanism of the collagen conformation in the environment of sodium sulphide (Na2S) solution is periodically studied by FT-IR spectroscopy, the Gaussian peak fitting of secondary derivative, and auxiliary methods such as SDS-PAGE, SEM and TG. As a result, the three polypeptide chains structure of bovine hide collagen was hardly affected by the ions of OH−, HS− and S2−. The conformation of collagen molecules such as β-turn, triple helix and β-strand were gradually evolved into unordered structure and α-helix with the sequential addition of OH−, HS− and S2−, which is because the hydrogen bond inside collagen molecular is extended or opened with the increase of the synergistic effect of pH and reducibility of HS− and S2− in the solution. The bound water content and thermal properties of the treated collagen molecules are decreased. Finally, the order of the hydrolysis ability to collagen is NaOH < NaHS < Na2S at the same condition, which was confirmed by measuring the content of hydroxyproline from their solutions after treating the collagen. This work provides very important information for understanding the evolutional mechanism of bovine hide collagen conformation in the Na2S solution, developing the alternatives of sulfide, extraction of collagen, tanning, and application of collagen in biological and material science.

The Enzyme Gene Expression of Protein Utilization and Metabolism by Lactobacillus helveticus CICC 22171.

The purpose of this study was to explore the hydrolytic ability of Lactobacillus helveticus CICC 22171 with regard to protein and the expression of enzyme genes during protein utilization. The results revealed that the strain hydrolyzed casein from the C-terminal, reached the maximum level in 6 h, and the number of amino acids in the hydrolyzed peptide was 7–33. The molecular weight was 652.4–3432.74 kDa. Hydrophobic peptides produced by hydrolysis were the source of β-casein bitterness. Leucine and glutamine were the preferred cleavage points after 1 h; tyrosine and tryptophan subsequently increased. The first step of hydrolysis was controlled by PrtP and PrtM genes and coordinated with the action of PrtH1 and PrtH2. The transport system consisted of DtpT, OppB, OppD and OppF. The hydrolytic third step endopeptidase system consisted of the aminopeptidases (PepN, PepC, PepM and PepA), the endopeptidases (PepE, PepF and PepO); the dipeptidases (PepV and PepD), the tripeptidase PepT; the proline peptidases (PepX, PepP, PepQ, PepR and PepI). The expression of CEP genes was significantly different, and the expression level of genes related to the transport system significantly increased from 0 to 1 h. The specificity of the substrate and action site of endopeptidase was abundant.

Deciphering the underlying mechanism of MOF-808-based abiotic catalysis enhancing biodegradability of waste activated sludge: Insights from the effects on bioconversion of extracellular organic substances into methane

Extracellular organic substances (EOSs) usually control sludge biodegradability. Metal−organic framework-808 (MOF-808), a catalyst with a high ability of catalytic hydrolysis and proton transfer, has great potential to enhance biodegradability of EOSs. In this study, the underlying mechanism of MOF-808 enhancing the biodegradability of EOSs via abiotic catalysis was investigated. Experimental results showed that protein–like and humic acid–like substances were the main organic components in EOSs, and the MOF-808 enhanced the disintegration of protein-like substances rather than humic acid–like substances. Analyses of the changes in the functional group, the secondary structure of protein-like substances, and the electron transfer of catalytically degraded EOS samples revealed that the MOF-808 enhanced the hydrolysis of EOSs protein-like substances, loosened their secondary structure, and improved the electron transfer in EOSs. Further analyses of the MOF-808 before and after the catalysis reaction revealed that the coordination of Zr sites with protein-like substance-specific active sites (such as ZrN) in the MOF-808 substantially contributed to the high-efficiency catalysis. Biochemical methane potential assays confirmed that MOF-808–based abiotic catalysis promoted the generation efficiency of methane from the EOSs. These findings can elucidate the abiotic catalytic effect of MOF-808 on sludge biodegradability during the anaerobic digestion process.

Production and Functional Characterization of a Novel Mannanase from Alteromonadaceae Bacterium Bs31.

Mannans are the main components of hemicellulose in nature and serve as the major storage polysaccharide in legume seeds. To mine new mannanase genes and identify their functional characteristics are an important basis for mannan biotechnological applications. In this study, a putative mannanase gene (ManBs31) from the genome of the marine bacterium Alteromonadaceae Bs31 was characterized. Amino acid sequence analysis and protein structural modeling were used to reveal the molecular features of ManBs31. The catalytic domain of ManBs31 was recombinantly produced using Escherichia coli and Pichia pastoris expression systems. The biochemical properties of the enzymes were determined by reducing sugar assay and thin-layer chromatography. Sequence analysis revealed that ManBs31 was a multidomain protein, consisting of a catalytic domain belonging to glycoside hydrolase family 5 (GH5) and two cellulose-binding domains. Recombinant ManBs31-GH5 exhibited the maximum hydrolytic performance at 70 ºC and pH 6. It showed the best hydrolysis capacity toward konjac glucomannan (specific enzyme activity up to 1070.84 U/mg) and poor hydrolysis ability toward galactomannan with high side-chain modifications (with a specific activity of 344.97 U/mg and 93.84 U/mg to locust bean gum and ivory nut mannan, respectively). The hydrolysis products of ManBs31-GH5 were mannooligosaccharides, and no monosaccharide was generated. Structural analysis suggested that ManBs31-GH5 had a noncanonical +2 subsite compared with other GH5 mannanases. ManBs31 was a novel thermophilic endo-mannanase and it provided a new alternative for the biodegradation of mannans, especially for preparation of probiotic mannooligosaccharides.

Probing strigolactone perception mechanisms with rationally designed small-molecule agonists stimulating germination of root parasitic weeds

The development of potent strigolactone (SL) agonists as suicidal germination inducers could be a useful strategy for controlling root parasitic weeds, but uncertainty about the SL perception mechanism impedes real progress. Here we describe small-molecule agonists that efficiently stimulate Phelipanchce aegyptiaca, and Striga hermonthica, germination in concentrations as low as 10−8 to 10−17 M. We show that full efficiency of synthetic SL agonists in triggering signaling through the Striga SL receptor, ShHTL7, depends on the receptor-catalyzed hydrolytic reaction of the agonists. Additionally, we reveal that the stereochemistry of synthetic SL analogs affects the hydrolytic ability of ShHTL7 by influencing the probability of the privileged conformations of ShHTL7. Importantly, an alternative ShHTL7-mediated hydrolysis mechanism, proceeding via nucleophilic attack of the NE2 atom of H246 to the 2′C of the D-ring, is reported. Together, our findings provide insight into SL hydrolysis and structure-perception mechanisms, and potent suicide germination stimulants, which would contribute to the elimination of the noxious parasitic weeds.

Characterization of a New Multifunctional GH20 β-N-Acetylglucosaminidase From Chitinibacter sp. GC72 and Its Application in Converting Chitin Into N-Acetyl Glucosamine.

In this study, a gene encoding β-N-acetylglucosaminidase, designated NAGaseA, was cloned from Chitinibacter sp. GC72 and subsequently functional expressed in Escherichia coli BL21 (DE3). NAGaseA contains a glycoside hydrolase family 20 catalytic domain that shows low identity with the corresponding domain of the well-characterized NAGases. The recombinant NAGaseA had a molecular mass of 92 kDa. Biochemical characterization of the purified NAGaseA revealed that the optimal reaction condition was at 40°C and pH 6.5, and exhibited great pH stability in the range of pH 6.5–9.5. The Vmax, Km, kcat, and kcat/Km of NAGaseA toward p-nitrophenyl-N-acetyl glucosaminide (pNP-GlcNAc) were 3333.33 μmol min–1 l–1, 39.99 μmol l–1, 4667.07 s–1, and 116.71 ml μmol–1 s–1, respectively. Analysis of the hydrolysis products of N-acetyl chitin oligosaccharides (N-Acetyl COSs) indicated that NAGaseA was capable of converting N-acetyl COSs ((GlcNAc)2–(GlcNAc)6) into GlcNAc with hydrolysis ability order: (GlcNAc)2 > (GlcNAc)3 > (GlcNAc)4 > (GlcNAc)5 > (GlcNAc)6. Moreover, NAGaseA could generate (GlcNAc)3–(GlcNAc)6 from (GlcNAc)2–(GlcNAc)5, respectively. These results showed that NAGaseA is a multifunctional NAGase with transglycosylation activity. In addition, significantly synergistic action was observed between NAGaseA and other sources of chitinases during hydrolysis of colloid chitin. Finally, 0.759, 0.481, and 0.986 g/l of GlcNAc with a purity of 96% were obtained using three different chitinase combinations, which were 1.61-, 2.36-, and 2.69-fold that of the GlcNAc production using the single chitinase. This observation indicated that NAGaseA could be a potential candidate enzyme in commercial GlcNAc production.

Potential Fungi Isolated From Anti-biodegradable Chinese Medicine Residue to Degrade Lignocellulose.

Traditional Chinese medicine is one of the ancient medicines which is popular in Asian countries, among which the residue produced by the use of anti-biodegradables is endless, and causes significant adverse impacts on the environment. However, the high acidity of anti-biodegradable residues and some special biological activities make it difficult for microorganisms to survive, resulting in a very low degradation rate of lignocellulose in naturally stacked residues, which directly impedes the degradation of residues. We aimed to identify the fungal strains that efficiently biodegrade anti-biodegradable residue and see the possibility to improve the biodegradation of it and other agricultural wastes by co-cultivating these fungi. We isolated 302 fungal strains from anti-biodegradable residue to test hydrolysis ability. Finally, we found Coniochaeta sp., Fomitopsis sp., Nemania sp., Talaromyces sp., Phaeophlebiopsis sp. which inhabit the anti-biodegradable residues are capable of producing higher concentrations of extracellular enzymes. Synergistic fungal combinations (viz., Fomitopsis sp. + Phaeophlebiopsis sp.; Talaromyces sp. + Coniochaeta sp. + Fomitopsis sp.; Talaromyces sp. + Fomitopsis sp. + Piloderma sp. and Talaromyces sp. + Nemania sp. + Piloderma sp.) have better overall degradation effect on lignocellulose. Therefore, these fungi and their combinations have strong potential to be further developed for bioremediation and biological enzyme industrial production.