High Hydrolytic Activity Research Articles

Production of N-Acetyl-d-glucosamine from Mycelial Waste by a Combination of Bacterial Chitinases and an Insect N-Acetyl-d-glucosaminidase.

N-Acetyl-d-glucosamine (GlcNAc) has great potential to be used as a food additive and medicine. The enzymatic degradation of chitin-containing biomass for producing GlcNAc is an eco-friendly approach but suffers from a high cost. The economical efficiency can be improved by both optimizing the member and ratio of the chitinolytic enzymes and using new inexpensive substrates. To address this, a novel combination of bacterial and insect chitinolytic enzymes was developed in this study to efficiently produce GlcNAc from the mycelia of Asperillus niger, a fermentation waste. This enzyme combination contained three bacterial chitinases (chitinase A from Serratia marcescens (SmChiA), SmChiB, SmChiC) and one insect N-acetyl-d-glucosaminidase from Ostrinia furnacalis (OfHex1) in a ratio of 39.1% of SmChiA, 26.7% of SmChiB, 32.9% of SmChiC, and 1.3% of OfHex1. A yield of 6.3 mM (1.4 mg/mL) GlcNAc with a purity of 95% can be obtained from 10 mg/mL mycelial powder in 24 h. The enzyme combination reported here exhibited 5.8-fold higher hydrolytic activity over the commercial chitinase preparation derived from Streptomyces griseus.

Purification and characterisation of a salt-stable protease from the halophilic archaeon Halogranum rubrum.

Because proteases play an important role in the fermentation of fish sauce, the purification and characterisation of an extracellular protease from the halophilic archaeon Halogranum rubrum was investigated. The molecular mass of the protease was estimated to be approximately 47 kDa based on sodium dodecyl sulfate-polyacrylamide gel electropheresis (SDS-PAGE) and native-PAGE analysis. The optimum conditions for catalytic activity were pH 8.0 and 50°C. The protease showed alkaline stability (pH 7.0-10.0). The protease also exhibited novel catalytic ability over a broad range of salinity (NaCl 0-3 mol L-1 ). Calcium ion enhanced the proteolytic activity of the enzyme. The Km and Vmax values of the purified protease for casein were calculated to be 4.89 mg mL-1 and 1111.11 U mL-1 , respectively. The protease was strongly inhibited by ethylenediamine tetraacetic acid (EDTA) and phenylmethanesulfonyl fluoride (PMSF). Meanwhile, the protease was stable in the presence of Triton X-100, isopropanol, ethanol or dithio-bis-nitrobenzoic (DTNB), but was inhibited by sodium dodecyl sulfate (SDS), dimethyl sulfoxide (DMSO) or methanol. MALDI -TOF/TOF MS analysis revealed that the protease shared some functional traits with protease produced by Halogranum salarium. Furthermore, it exhibited high hydrolytic activity on silver carp myosin protein. The protease is an alkaline and salt-tolerant enzyme that hydrolyses silver carp myosin with high efficiency. These excellent characteristics make this protease an attractive candidate for industrial use in low-salt fish sauce fermentation. © 2016 Society of Chemical Industry.

Effects of biocide treatments on the biofilm community in Domitilla's catacombs in Rome

Different types of biofilms are widespread on lithic faces of the Catacombs of Domitilla (Rome, Italy) due to the favorable microclimatic conditions (temperature, high RH% and low irradiance). The biofilm, once established, becomes particularly dangerous due to the coverage of valuable surfaces causing spoilage, softening of materials and mineral precipitation. It is common practice to treat these surfaces with biocides in order to eradicate the microorganisms present. The aim of the present research was to compare the changes occurring to the microbial community present in the biofilm in one site of the Catacombs of Domitilla (CD15) before and after a biocide treatment (a mixture of quaternary ammonium compounds and octylisothiazolone, OIT), applied for a one month period. A multistep approach was followed, based on microscopy, cultural methods and molecular techniques (f-ITS and 16S rDNA sequencing), for the phenotypic and genetic analysis of the culturable microbial population. Our results highlighted that the biocide treatments had little effect against cyanobacteria, while the bacterial population increased in numbers but changed drastically in terms of diversity. In fact, some bacteria proliferate at the expense of the organic matter released by dead microorganisms as demonstrated by laboratory tests. Further, our data describe how the microbial interaction can have different responses depending on the favorable conditions for one kind of microorganism in respect to the others.This study exemplifies the real risks of applying biocide treatments on complex microbial communities and pinpoints the necessity of subjecting treatments to monitoring and reassessment. Moreover, the work showed the potential of bacteria isolated after the treatment for use, under controlled conditions, in combatting unwanted microbial growth in that they possess a positive tropism toward stressed microorganisms and high hydrolytic enzymatic activity against cell components (e.g. cellulose, chitin and pectin). A tentative protocol is proposed.

Enzymatic in situ saccharification of lignocellulosic biomass in ionic liquids using an ionic liquid-tolerant cellulases

Ionic liquids (ILs) have been considered as a class of promising solvents that can dissolve lignocellulosic biomass and then provide enzymatic hydrolyzable holocellulose. However, most of available cellulases are completely or partially inactivated in even low concentrations of ILs. To more fully exploit the benefits of ILs to lignocellulose biorefinery, it is critical to improve the compatibility between cellulase and ILs. In this study, an IL-tolerant cellulase cocktail derived from Penicillium oxalicum GS was isolated from chemicals polluted microhabitats. High hydrolytic activity of the cellulase cocktail was detected in various ILs. In particular, the cellulase cocktail retained 91% initial activity in IL dimethylimidazolium methylphosphonate ([Dmim][(OCH3)2PO2]) (20%, w/v), which was effective in disrupting compact structures of rice straw. Furthermore, the cellulase cocktail even preserved 61% initial activity in 25% (w/v) [Dmim][(OCH3)2PO2] media. A compatible cellulase-IL system, which combines IL pretreatment and subsequent enzymatic saccharification of lignocellulosic biomass into a single vessel, was then developed based on the cellulase cocktail and [Dmim][(OCH3)2PO2]. Its use significantly improved the saccharification rate of rice straw from 47% to 83% versus the control.

The Vibrio parahaemolyticus-infecting bacteriophage qdvp001: genome sequence and endolysin with a modular structure.

Vibrio parahaemolyticus, a marine pathogen, is a causative agent of gastroenteritis in humans after consumption of contaminated seafood. In recent years, infections with V. parahaemolyticus have become an increasingly frequent factor in microbial food poisoning; therefore, it is urgent to figure out ways to control Vibrio parahaemolyticus. Endolysins, lytic enzymes encoded by bacteriophages, have been regarded as a therapeutic alternative to antibiotics in control of bacterial growth and have been successfully utilized in various areas. Here, we report the full genome sequence of the novel phage qdvp001, which lyses Vibrio parahaemolyticus 17802. The qdvp001 genome consists of a 134,742-bp DNA with a G+C content of 35.35% and 227 putative open reading frames. Analysis revealed that the qdvp001 open reading frames encoded various putative functional proteins with a putative endolysin gene (ORF 60). No holin genes were identified in qdvp001. ORF 60 was cloned and expressed. The results showed that the purified endolysin Lysqdvp001 had a high hydrolytic activity toward Vibrio parahaemolyticus and a broader spectrum compared to that of the parental bacteriophage qdvp001. Thus, purified endolysin Lysqdvp001 has a potential to be used as an antibacterial agent in the future.

Magnetic field induced synthesis of amorphous CoB alloy nanowires as a highly active catalyst for hydrogen generation from ammonia borane

CoB nanowires are prepared by a facile process free of any template or capping agent. The cleanly-surfaced CoB nanowires exhibit high catalytic activity in hydrolysis of ammonia borane, and the turnover frequency value is 4.88LH2min−1gcatalyst−1, which is significantly higher than those of recently reported CoB nanocatalysts. The apparent activation energy of catalytic hydrolysis of ammonia borane is as low as 16.2kJmol−1, hinting the high catalytic activity of the CoB nanowires. The high catalytic performance and good recoverability make the CoB nanowires a strong catalyst candidate in the hydrolysis of ammonia borane for hydrogen generation.

Enhancing tungsten oxide/SBA-15 catalysts for hydrolysis of cellobiose through doping ZrO2

The number of strong acid sites on a hydrolysis catalyst accessible by the bulky cellulose limits its efficiency in the conversion and utilization of biomass-based resources. In this work, high surface area WZr/SBA-15 catalysts (>400m2/g) were prepared by hydrothermal method and characterized using XRD, N2 adsorption, TEM, Raman, UV–vis, XPS and FTIR of pyridine adsorption. The results showed that doping a moderate amount of ZrO2 into the WO3/SBA-15 catalyst stabilized the tungstate species in the form of a polytungstate species as a result of the strong ZrOx-WOx interaction as well as increased the surface area of the catalyst. The resulting polytungstate species provided strong Brønsted acid sites for hydrolysis. Consequently, the cellobiose conversion was maximized at 5% ZrO2 loading, corresponding to a maximized surface area and density of Brønsted acid sites. This catalytic activity is significantly higher than that of a WO3/ZrO2 catalyst prepared using the conventional impregnation method, indicating the importance of mesoporous structure to the dispersion of active species and to the accessibility of the active sites by the bulky reactant. This result indicates that the density of the strong Brønsted acid sites with improved accessibility by the bulky reactant is essential to achieve a high hydrolysis activity.

Immobilization of Candida antarctica lipase B onto SBA-15 and their application in glycerolysis for diacylglycerols synthesis

In this study, Candida antarctica lipase B (CALB) was immobilized on SBA-15 with three pore diameters. CALB loading was found increased with CALB concentration increasing from 20.3 to 80.12μg/ml. Higher CALB loading was observed from SBA-15 with pore diameters at 8.1nm (SBA-15(8.1)), yet highest hydrolytic activity was found at SBA-15(12.5). Thermal stability of the immobilized CALB (SBA-15-CALB) samples was greatly influenced by their water content, after 4h storage at 70°C, 8.93 and 67.4% of the initial activity was observed from SBA-15-CALB samples with water content at 9.23 and 3.22% respectively. The SBA-15-CALB samples were then used in glycerolysis of corn oil, and 53.6wt% of diacylglycerols was obtained after optimization. The operational stability was tested, and after 5 consecutive applications, 92.5 and 80.3% of the initial glycerolysis activity was remained respectively from SBA-15(6.6)-CALB and SBA-15(12.5)-CALB.

The efficiency of synthesis of β-fructofuranosidase by Debaryomyces hansenii yeast when cultured on a nutrient medium from molasses

Distribution D. hansenii in different substrates can be explained sufficiently high hydrolytic activity, which is a prerequisite for their use in biotechnology to produce various biological products, in particular lytic enzymes. The paper is picked productivity and efficiency of synthesis of extracellular enzyme β-fructofuranosidase D. hansenii yeast nutrient medium from molasses. The efficiency of D. hansenii yeast culture in a nutrient medium of molasses depends primarily on the strain. It was found that the most productive and efficient synthesizing extracellular enzyme β-fructofuranosidase is yeast strain D. hansenii H4651. Maximum productivity and efficiency of the synthesis of the extracellular enzyme β-fructofuranosidase D. hansenii yeast strain H4651 seen when cultured in a nutrient medium from molasses without additional nutrients at a temperature of 20 oC. It was found that the consumption of reducing substances is not an adequate amount of yeast biomass synthesized D. hansenii. The content of reducing substances after 50 hours of cultivation D. hansenii yeast H4651 Uwe-creases. The resulting pattern PB increase in liquid culture may explain synthesis drozh-Jamie D. hansenii β – fructofuranosidase extracellular enzyme which hydrolyzes sucrose medium. The highest specific growth rate observed in yeast D. hansenii H4651 at temperatures cultivated-tion 15 and 20 ° C. Out D. hansenii yeast biomass H4651 exceeds the yield of biomass when cultured yeast D. hansenii strains H433 and H18–3.

Derivatization of Proteinase K with Heavy Atoms Enhances Its Thermal Stability

Proteinase K is widely used in molecular biology for its broad substrate specificity, wide pH stability, and high hydrolysis activity. Aminolysis by proteinase K is also attractive for chemoenzymatic peptide synthesis. However, the relatively low thermal stability of the native protease limits the hydrolysis and aminolysis reactions in industrial processes. Here, we enhanced the thermal stability of proteinase K using a heavy-atom derivatization technique. The denaturation temperature of proteinase K derivatized with praseodymium (Pr) ions was 16.2 and 5.9 °C higher than those of metal-free and Ca-binding proteinase K, respectively. Isothermal titration calorimetry (ITC) measurements demonstrated that Pr-ion binding to proteinase K showed endothermic peaks, whereas Ca-ion binding showed exothermic peaks, indicating that the binding mode of Pr ions was different from that of Ca ions, even though the crystal structures of proteinase K with Pr and Ca ions were identical. Hydrolytic activity of Pr-derivatized...

Characterization of a Lignocellulolytic Consortium and Methane Production from Untreated Wheat Straw: Dependence on Nitrogen and Phosphorous Content

Impacts of microbial diversity and macronutrients levels (expressed as C:N and C:P ratios) on the methane production from an untreated lignocellulosic feedstock were assessed. Next-generation sequencing technology revealed the bacterial diversity of a lignocellulolytic inoculum. This inoculum comprised 75 bacterial species that were well distributed in 14 phyla, 67% of which belonged to Firmicutes and Bacteroidetes. The families Ruminococcaceae, Clostridiaceae, Bacteroidaceae, Bacillaceae, and Fibrobacteraceae comprised 46% of the identified families and were associated with hydrolytic members. Nutrient adjustment reduced 40% of the length of the lag phase and doubled methane production rate compared with a control. The highest methane production of 0.197 m3 per kg of total volatile solids observed at C:N of 31:1 and C:P of 428:1, peaked 20 days earlier than in previous studies using untreated lignocellulosic feedstock. Interestingly, the highest hydrolytic activities and solids removal rates were observed at high nitrogen contents; however, the conditions (pH > 8.0) inhibited methanogenesis.

Lipase cocktail for efficient conversion of oils containing phospholipids to biodiesel

The presence of phospholipid has been a challenge in liquid enzymatic biodiesel production. Among six lipases that were screened, lipase AY had the highest hydrolysis activity and a competitive transesterification activity. However, it yielded only 21.1% FAME from oil containing phospholipids. By replacing portions of these lipases with a more robust bioFAME lipase, CalT, the combination of lipase AY–CalT gave the highest FAME yield with the least amounts of free fatty acids and partial glycerides. A higher methanol addition rate reduced FAME yields for lipase DF–CalT and A10D–CalT combinations while that of lipase AY–CalT combination improved. Optimizing the methanol addition rate for lipase AY–CalT resulted in a FAME yield of 88.1% at 2h and more than 95% at 6h. This effective use of lipases could be applied for the rapid and economic conversion of unrefined oils to biodiesel.

Development of a bifunctional catalyst for dimethyl ether steam reforming with CuFe2O4 spinel as the metallic function

This study aims at optimizing the composition of the bifunctional catalyst with CuFe2O4 spinel metallic function for the steam reforming of DME (SRD). The γ-Al2O3 obtained by calcination of boehmite at 550°C proves to be the most suitable acid function, given that it provides high activity for DME hydrolysis and avoids hydrocarbon formation in the 300–400°C range. Furthermore, the optimum mass ratio between CuFe2O4 and γ-Al2O3 functions is 1/1 at 350°C, since it allows obtaining the highest DME conversion and H2 yield with suitable catalyst stability and mechanical resistance.

Evaluation of wild herbivore faeces from South Africa as a potential source of hydrolytically active microorganisms.

This study assessed faecal matter from three indigenous South African herbivores—zebra, giraffe and impala—as a potential source for hydrolytically active aerobic and facultatively anaerobic bacteria. Herbivore droppings were collected freshly in a local nature reserve in Pietermaritzburg, South Africa. Soil samples adjacent to faecal collection sites and faeces from a domestic herbivore, the Nguni cow, were included as controls. Hydrolase and dehydrogenase activity in faecal matter and soil samples were measured by the fluorescein diacetate and the triphenyltetrazolium chloride assay. Viable counts and counts for amylase, cellulase, esterase and protease producers were established using plate count agar and solid media containing cellulose, skim milk, starch and Tween 80. Zebra droppings produced the highest hydrolase and dehydrogenase activity. Faecal matter of the three indigenous herbivores generally produced higher hydrolytic activity than Nguni cow faeces and soil controls, thereby confirming that these materials are potential targets for hydrolytic enzyme mining.

Cloning, Purification, and Characterization of a Heterodimeric β-Galactosidase from Lactobacillus kefiranofaciens ZW3.

Lactobacillus kefiranofaciens ZW3 was obtained from kefir grains, which have high lactose hydrolytic activity. In this study, a heterodimeric LacLM-type β-galactosidase gene (lacLM) from ZW3 was isolated, which was composed of two overlapping genes, lacL (1,884 bp) and lacM (960 bp) encoding large and small subunits with calculated molecular masses of 73,620 and 35,682 Da, respectively. LacLM, LacL, and LacM were expressed in Escherichia coli BL21(DE3) and these recombinant proteins were purified and characterized. The results showed that, compared with the recombinant holoenzyme, the recombinant large subunit exhibits obviously lower thermostability and hydrolytic activity. Moreover, the optimal temperature and pH of the holoenzyme and large subunit are 60°C and 7.0, and 50°C and 8.0, respectively. However, the recombinant small subunit alone has no activity. Interestingly, the activity and thermostability of the large subunit were greatly improved after mixing it with the recombinant small subunit. Therefore, the results suggest that the small subunit might play an important role in maintaining the stability of the structure of the catalytic center located in the large subunit.

Facile synthesis of novel Co-B@Ni/RGO nanocomposite: A cost effective catalyst for improved hydrogen generation with enhanced electrochemical activity

Novel catalyst, cobalt boride @ nickel/reduced graphene oxide abbreviated as Co-B@Ni/RGO nanocomposite was synthesized by precipitation of Co2+ ions on Ni/RGO. In the first step, Ni/RGO nanocomposite was synthesized by a facile method. In the next step, Co-B NPs were decorated on Ni/RGO support which led the superficial attachment and maximum exposure of them for catalytic reaction. The structure, composition and morphology of catalyst were fully characterized using various techniques. Co-B@Ni/RGO nanocomposite showed the high catalytic activity for hydrolysis of alkaline sodium borohydride (NaBH4) solution. Moreover, Co-B@Ni/RGO nanocomposite showed the high current response and stability of catalyst under cyclic voltammetry (CV) experiment, suggested as promising low cost nanocatalyst for future energy applications due to its high catalytic activity, charge storage capability and robustness.

Accessing regio-and typo-selectivity of Yarrowia lipolytica lipase in its free form and immobilized onto magnetic nanoparticles

The growing interest in lipases is related to its high biotechnological potential. In this work we investigated the regio- and typo-selectivity of extracellular lipase from Yarrowia lipolytica 583 (IMUFRJ 50682) in its free form and immobilized on magnetic nano-sized particles. Results of p-nitrophenyl laurate hydrolysis showed optimal stability of these biocatalysts at pH 6–8 and between 20 and 45°C. The immobilization of lipase on nano-sized magnetic particles increased thermo and pH stabilities of the enzyme. Both catalysts displayed high hydrolytic activity on triolein and on triacylglycerols in vegetable oils in 5min. The free lipase showed a strict 1,3-regioselectivity, whereas the immobilized lipase was only slightly 1,3-regioselective, in hydrolysis reactions of triolein and vegetable oils. Both lipases were able to hydrolyze saturated esters, but showed no typo-selectivity for this group. However, the lipases are selective for the hydrolysis of unsaturated esters, especially for 18:2 fatty acids. These results give a better knowledge of this lipase hydrolytic activity, both in the free form and immobilized and widen their potential biotechnological use, especially in the synthesis of structured lipids.

Epoxy-silanization - tool for improvement of silica nanoparticles as support for lipase immobilization with respect to esterification activity

BACKGROUND In this study, in order to obtain the best support for the immobilization of lipase from Candida rugosa (CRL), the procedure for chemical modification of fumed silica nanoparticles (FNS) with (3-Glycidyloxypropyl)trimethoxysilane (GOPTMS) was optimized by varying the amount of GOPTMS used and the duration of support modification. To evaluate the prospects of chemical modification of FNS surface on the immobilization process, the activity and thermal stability of lipase immobilized on GFNS was compared with lipase immobilized on FNS and cyanuric chloride functionalized amino silica (CCAFNS). RESULTS The epoxy functionalized silica nanoparticles (GFNS) with 400–500 µmol of introduced epoxy groups per g of support were the most efficient, since immobilized preparations in 1 mol L−1 buffer had the highest activity of approximately 1495 IU g−1 and a loading capacity of 294 mg g−1. Lipase immobilized on GFNS exhibited 1.5-fold higher hydrolytic activity and 2-fold higher esterification activity in the synthesis of aroma esters than lipase immobilized on CCAFNS, while its thermal stability was similar to CCAFNS preparations. CONCLUSION These results indicated that the type of modification of the FNS surface has an influence on the specificity of the immobilized lipase, since lipase immobilized on GFNS showed improved properties for ester synthesis which is a promising area for immobilized lipase application. © 2015 Society of Chemical Industry

Characterization of a novel cold active and salt tolerant esterase from Zunongwangia profunda

A novel cold active esterase, EstLiu was cloned from the marine bacterium Zunongwangia profunda, overexpressed in E. coli BL21 (DE3) and purified by glutathione-S transferase (GST) affinity chromatography. The mature esterase EstLiu sequence encodes a protein of 273 amino acids residues, with a predicted molecular weight of 30KDa and containing the classical pentapeptidase motif from position 156 to 160 with the catalytic triad Ser158-Asp211-His243. Although, EstLiu showed 64% similarity with the hypothetical esterase from Chryseobacterium sp. StRB126 (WP_045498424), phylogenetic analysis showed it had no similarity with any of the established family of lipases/esterases, suggesting that it could be considered as a new family. The purified enzyme showed broad substrate specificity with the highest hydrolytic activity against p-nitrophenyl butyrate (C4). EstLiu showed remarkable activity (75%) at 0°Cand the optimal activity at pH 8.0 and 30°C with good thermostability and quickened inactivation above 60°C. EstLiu retained 81, 103, 67 and 78% of its original activity at 50% (v/v) in ethanol, isopropanol, DMSO and ethylene glycol, respectively. In the presence of Tween 20, Tween 80 and Triton X-100, EstLiu showed 88, 100 and 117% of relative activity. It is also co-factor independent. The high activity at low temperature and desirable stability in organic solvents and salts of this novel family esterase represents a good evidence of novel biocatalyst. Overall, this novel enzyme showed better activity than previously reported esterases in extreme reaction conditions and could promote the reaction in both aqueous and non-aqueous conditions, indicating its great potential for industrial applications.

Characteristics of Sigumjang, fermented barley bran paste, usually consumed in Gyeongsang-do Area of South Korea and Isolation of its useful Strains

Sigumjang made from barley bran is a traditional fermented food only found in Gyungsang-do area of South Korea. Appearance, tastes and flavors of sigumlang differs from Doenjang (well-known Korean traditional fermented soybean paste). We investigated characteristics of sigumjang and isolated strains of microorganism with high enzymatic activity from sigumjang meju. We collected 19 kinds of sigumjang and 14 kinds of sigumjang meju, which is dried-fermented barley bran and serves as the basis of sigumjang. Methods of sigumjang meju-making are different by region from baking directly the dough of barley bran or after steaming the dough. Sigumjang showed lower pH of 4.37 - 5.99 compared to other Korean traditional fermented jang, because of its lactic acid fermentation. 13 strains showed high hydrolysis activity of cellulose, starch, protein among 51 strains collected from the 14 kinds of sigumjang meju. SM-26 stain showed a high enzymatic activity of cellulase, amylase and protease. SM-26 stain was identified as Bacillus amyloliquefaciens subsp. based on its 16S rRNA sequence analysis.