Synthesis of urease hybrid nanoflowers and their enhanced catalytic properties

Increasing numbers of materials have been extensively used as platforms for enzyme immobilization to enhance catalytic activity and stability. Although stability of enzyme was accomplished with immobilization approaches, activity of the most of the enzymes was declined after immobilization. Herein, we synthesize the flower shaped-hybrid nanomaterials called hybrid nanoflower (HNF) consisting of urease enzyme and copper ions (Cu²⁺) and report a mechanistic elucidation of enhancement in both activity and stability of the HNF. We demonstrated how experimental factors influence morphology of the HNF. We proved that the HNF (synthesized from 0.02 mg mL⁻¹ urease in 10 mM PBS (pH 7.4) at +4 °C) exhibited the highest catalytic activity of ∼2000% and ∼4000% when stored at +4 °C and RT, respectively compared to free urease. The highest stability was also achieved by this HNF by maintaining 96.3% and 90.28% of its initial activity within storage of 30 days at +4 °C and RT, respectively. This dramatically enhanced activity is attributed to high surface area, nanoscale-entrapped urease and favorable urease conformation of the HNF. The exceptional catalytic activity and stability properties of HNF can be taken advantage of to use it in fields of biomedicine and chemistry.     

Application of chitosan beads immobilized Rhodococcus sp. NCIM 2891 cholesterol oxidase for cholestenone production

The cell-bound cholesterol oxidase from the Rhodococcus sp. NCIM 2891 was purified three fold by diethylaminoethyl–sepharose chromatography. The estimated molecular mass (SDS-PAGE) and K_m of the enzyme were ∼55.0 kDa and 151 μM, respectively. The purified cholesterol oxidase was immobilized on chitosan beads by glutaraldehyde cross-linking reaction and immobilization was confirmed by Fourier transform infrared spectroscopy, scanning electron microscopy and energy dispersive X-ray analysis. The optimum temperature (45 °C, 5 min) for activity of the enzyme was increased by 5 °C after immobilization. Both the free and immobilized cholesterol oxidases were found to be stable in many organic solvents except for acetone. Fe²⁺ and Pb²⁺ at 0.1 mM of each acted as inhibitors, while Ag⁺, Ca²⁺, Ni²⁺ and Zn²⁺ activated the enzyme at similar concentration. The biotransformation of cholesterol (3.75 mM) with the cholesterol oxidase immobilized beads (3.50 U) leads to ∼88% millimolar yield of cholestenone in a reaction time of 9 h at 25 °C. The immobilized enzyme retains ∼67% activity even after 12 successive batches of operation. The biotransformation method thus, shows a great promise for the production of pharmaceutically important cholestenone.     

Purification of a laccase from fungus combs in the nest of Odontotermes formosanus

A new enzyme was isolated from the fungus combs in the nest of Odontotermes formosanus and identified as a laccase. The single laccase was purified with a purification factor of 16.83 by ammonium sulphate precipitation and anion exchange chromatography, to a specific activity of 211.11 U mg⁻¹. Its molecular mass was 65 kDa. The optimum pH value and temperature were 4.0 °C and 10 °C with ABTS as the substrate, respectively. The enzyme activity stabilized at temperatures between 10 °C and 30 °C and decreased rapidly when the temperature was above 30 °C. The V_max and K_m values were 3.62 μmol min⁻¹ mg⁻¹ and 119.52 μM, respectively. Ethanol concentration affected laccase activity, inhibiting 60% of enzyme activity at a concentration of 70%. Metal ions of Mg²⁺, Ba²⁺ and Fe²⁺ showed inhibition on enzyme activity of 17.2%, 5.3% and 9.4%, respectively, with the increase of metal ions concentration from 1 mM to 5 mM. Especially Fe²⁺ strongly inhibited enzyme activity up to 89% inhibition at a concentration of 1 mM.     

Stability and structural changes of horseradish peroxidase: Microwave versus conventional heating treatment

Effects of conventional heating (CH) and microwave (MW) on the structure and activity of horseradish peroxidase (HRP) in buffer solution were studied. CH incubation between 30 and 45 °C increased activity of HRP, reaching 170% of residual activity (RA) after 4–6 h at 45 °C. CH treatment at 50 and 60 °C caused HRP inactivation: RA was 5.7 and 16.7% after 12 h, respectively. Secondary and tertiary HRP structural changes were analyzed by circular dichroism (CD) and intrinsic fluorescence emission, respectively. Under CH, activation of the enzyme was attributed to conformational changes in secondary and tertiary structures. MW treatment had significant effects on the residual activity of HRP. MW treatment at 45 °C/30 W followed by CH treatment 45 °C regenerated the enzyme activity. The greatest loss in activity occurred at 60 °C/60 W/30 min (RA 16.9%); without recovery of the original activity. The inactivation of MW-treated HRP was related to the loss of tertiary structure, indicating changes around the tryptophan environment.     

Purification and characterization of two agarases from Agarivorans albus OAY02

Two extracellular β-agarases were purified from Agarivorans albus OAY2, which was isolated from seaweed collected in Qingdao, Shandong, China. The fermentation process was optimized with enzyme activity improving from 1.06 U/ml to 2.65 U/ml. Agarases were purified by ammonium sulfate fractionation and ion-exchange chromatography. Purification resulted in a 107-fold increase in agarase-a activity and a 52-fold increase in case of agarase-b, with high specific activities of 2715 and 1338 U/mg, respectively. The molecular masses of the agarases were estimated to be 50 kDa and 107 kDa, respectively. The optimum temperatures for the activities of agarase-a and agarase-b were 40 °C and 50 °C, respectively. Agarase-a was stable at 30 °C, while agarase-b was stable at 50 °C. The optimum pH values for agarase-a and agarase-b were both 9.0. Agarase-a was inhibited by Cu²⁺, Mn²⁺, Co²⁺ and EDTA, while agarase-b was inhibited by Cu²⁺, Mn²⁺ and Fe²⁺. The results of ¹³C NMR and TLC showed that hydrolysis of agarose by agarase-a produced neoagarobiose (NA2), neoagarotetraose (NA4) and neoagarohexaose (NA6). Agarase-b hydrolyzed agarose to yield neoagarobiose (NA2) and neoagarotetraose (NA4). Evaluation of matrix-assisted laser desorption ionization/time-of-flight MS (MALDI-TOF-TOF/MS) and the enzymatic product results indicated that agarase-a and agarase-b might be novel agarases.     

Multiobjective optimization of the antioxidant activities of horse mackerel hydrolysates produced with protease mixtures

Fish protein hydrolysates (FPH) from horse mackerel were produced by employing an enzyme mixture of subtilisin and trypsin. The antioxidant activity of fish hydrolysates (DPPH scavenging activity, Fe²⁺ chelating activity and Fe³⁺ reducing power) was modelled as a function of the operating conditions for the hydrolysis (i.e. protein concentration, temperature and composition of the enzyme mixture). The antioxidant activities showed different behavior depending on whether their controlling pathway was the transference of electrons/protons (i.e. DPPH scavenging activity and Fe³⁺ reducing power) or metal chelation. In the first case, the antioxidant activities increased with the decrease of substrate concentration and temperature when pure trypsin (DPPH scavenging activity) or a mixture of enzymes (Fe³⁺ reducing capacity) was employed. Contrarily, hydrolysates showed higher Fe²⁺ chelating activities at moderate concentration and high temperature (i.e. 5 g/L and 55 °C) employing solely subtilisin. The conflictive behavior among the antioxidant properties suggested using a multiobjective optimization technique. The ε-constraint method was chosen for this purpose. This approach allows determining the most adequate operational conditions for producing hydrolysate with a specific antioxidant profile which is the first approximation to the production of taylor-made antioxidant hydrolysates.     

Purification and biochemical properties of an extracellular acid phytase produced by the Saccharomyces cerevisiae CY strain

An extracellular acid phytase was purified to homogeneity from the culture supernatant of the Saccharomyces cerevisiae CY strain by ultrafiltration, DEAE-Sepharose column chromatography, and Sephacryl S-300 gel filtration. The molecular weight of the purified enzyme was estimated to be 630 kDa by gel filtration. Removing the sugar chain by endoglycosidase H digestion revealed that the molecular mass of the protein decreased to 446 kDa by gel filtration and gave a band of 55 kDa by SDS-PAGE. The purified enzyme was most active at pH 3.6 and 40 °C and was fairly stable from pH 2.5 to 5.0. The phytase displayed broad substrate specificity and had a K_m value of 0.66 mM (sodium phytate, pH 3.6, 40 °C). The phytase activity was completely inhibited by Fe³⁺ and Hg²⁺, and strongly inhibited (maximum of 91%) by Ba²⁺, Co²⁺, Cu⁺, Cu²⁺, Fe²⁺, Mg²⁺, and Sn²⁺ at 5 mM concentrations.     

Improving the catalytic performance of porcine pancreatic lipase in the presence of [MMIm][MeSO4] with the modification of functional ionic liquids

Porcine pancreatic lipase (PPL) was chemically modified with various functional ionic liquids (ILs) to increase its catalytic performance in water-miscible IL. Catalytic activity and thermostability were tested with a p-nitrophenyl palmitate (pNPP) hydrolysis reaction. The native enzyme lost 18% of its initial activity in 0.4 M [MMIm][MeSO₄], whereas the activities of all the modified enzymes increased. The [HOOCBMIm][Cl] modification led to a 2-fold increase in activity in 0.3 M [MMIm][MeSO₄] than in aqueous. All the modified enzymes exhibited higher thermostability compared with the native enzyme at high temperature. In particular, the [HOOCBMIm][Cl] modification led to a 6-fold increase in thermostability at 60 °C. Conformational changes were confirmed by fluorescence spectroscopy and circular dichroism spectroscopy to elucidate the mechanism of catalytic performance alteration.     

Purification and physicochemical properties of polygalacturonase from Aspergillus niger MTCC 3323

Polygalacturonases are the pectinolytic enzymes that catalyze the hydrolytic cleavage of the polygalacturonic acid chain. In the present study, polygalacturonase from Aspergillus niger (MTCC 3323) was purified. The enzyme precipitated with 60% ethanol resulted in 1.68-fold purification. The enzyme was purified to 6.52-fold by Sephacryl S-200 gel-filtration chromatography. On SDS–PAGE analysis, enzyme was found to be a heterodimer of 34 and 69 kDa subunit. Homogeneity of the enzyme was checked by NATIVE-PAGE and its molecular weight was found to be 106 kDa. The purified enzyme showed maximum activity in the presence of polygalacturonic acid at temperature of 45 °C, pH of 4.8, reaction time of 15 min. The enzyme was stable within the pH range of 4.0–5.5 for 1 h. At 4 °C it retained 50% activity after 108 h but at room temperature it lost its 50% activity after 3 h. The addition of Mn²⁺, K⁺, Zn²⁺, Ca²⁺ and Al³⁺ inhibited the enzyme activity; it increased in the presence of Mg²⁺ and Cu²⁺ ions. Enzyme activity was increased on increasing the substrate concentration from 0.1% to 0.5%. The K_m and V_max values of the enzyme were found to be 0.083 mg/ml and 18.21 μmol/ml/min. The enzyme was used for guava juice extraction and clarification. The recovery of juice of enzymatically treated pulp increased from 6% to 23%. Addition of purified enzyme increased the %T₆₅₀ from 2.5 to 20.4 and °Brix from 1.9 to 4.8. The pH of the enzyme treated juice decreased from 4.5 to 3.02.     

Immobilization of catalase onto Eupergit C and its characterization

Bovine liver catalase was covalently immobilized onto Eupergit C. Optimum conditions of immobilization: pH, buffer concentration, temperature, coupling time and initial catalase amount per gram of carrier were determined as 7.5, 1.0 M, 25 °C, 24 h and 4.0 mg/g, respectively. V_max and K_m were determined as 1.4(±0.2) × 10⁵ U/mg protein and 28.6 ± 3.6 mM, respectively, for free catalase, and as 3.7(±0.4) × 10³ U/mg protein and 95.9 ± 0.6 mM, respectively, for immobilized catalase. The thermal stability of the immobilized catalase in terms of half-life time (29.1 h) was comparably higher than that of the free catalase (9.0 h) at 40 °C. Comparison of storage stabilities showed that the free catalase completely lost its activity at the end of 11 days both at room temperature and 5 °C. However, immobilized catalase retained 68% of its initial activity when stored at room temperature and 79% of its initial activity when stored at 5 °C at the end of 28 days. The highest reuse number of immobilized catalase was 22 cycles of batch operation when 40 mg of immobilized catalase loaded into the reactor retaining about 50% of its original activity. In the plug flow type reactor, the longest operation time was found as 82 min at a substrate flow rate of 2.3 mL/min when the remaining activity of 40 mg immobilized catalase was about 50% of its original activity. The resulting immobilized catalase onto Eupergit C has good reusability, thermal stability and long-term storage stability.     

Comparative biochemical characterization of soluble and chitosan immobilized β-galactosidase from Kluyveromyces lactis NRRL Y1564

An investigation was conducted on the production of β-galactosidase (β-gal) by different strains of Kluyveromyces, using lactose as a carbon source. The maximum enzymatic activity of 3.8 ± 0.2 U/mL was achieved by using Kluyveromyces lactis strain NRRL Y1564 after 28 h of fermentation at 180 rpm and 30 °C. β-gal was then immobilized onto chitosan and characterized based on its optimal operation pH and temperature, its thermal stability and its kinetic parameters (K_m and V_max) using o-nitrophenyl β-d-galactopyranoside as substrate. The optimal pH for soluble β-gal activity was found to be 6.5 while the optimal pH for immobilized β-gal activity was found to be 7.0, while the optimal operating temperatures were 50 °C and 37 °C, respectively. At 50 °C, the immobilized enzyme showed an increased thermal stability, being 8 times more stable than the soluble enzyme. The immobilized enzyme was reused for 10 cycles, showing stability since it retained more than 70% of its initial activity. The immobilized enzyme retained 100% of its initial activity when it was stored at 4 °C and pH 7.0 for 93 days. The soluble β-gal lost 9.4% of its initial activity when it was stored at the same conditions.     

Thermophilic lipase from Thermomyces lanuginosus: Gene cloning,expression and characterization

An extracellular lipase gene ln1 from thermophilic fungus Thermomyces lanuginosus HSAUP₀₃80006 was cloned through RT-PCR and RACE amplification. Its coding sequence predicted a 292 residues protein with a 17 amino acids signal peptide. The deduced amino acids showed 78.4% similarity to another lipase lgy from T. lanuginosus while shared low similarity with other fungi lipases. Higher frequencies hydrophobic amino acids related to lipase thermal stability, such as Ala, Val, Leu and Gly were observed in this lipase (named LN). The sequence, -Gly-His-Ser-Leu-Gly-, known as a lipase-specific consensus sequence of mould, was also found in LN. High level expression for recombinant lipase was achieved in Pichia pastoris GS115 under the control of strong AOX1 promoter. It was purified to homogeneity through only one step DEAE-Sepharose anion exchange chromatography and got activity of 1328 U/ml. The molecular mass of one single band of this lipase was estimated to be 33 kDa by SDS-PAGE. The enzyme was stable at 60 °C and kept 65% enzyme activity after 30 min incubation at 70 °C. It kept half-activity after incubated for 40 min at 80 °C. The optimum pH for enzyme activity was 9.0 and the lipase was stable from pH 8.0 to 12.0. Lipase activity was enhanced by Ca²⁺ and inhibited by Fe²⁺, Zn²⁺, K⁺, and Ag⁺. The cell-free enzyme hydrolyzed and synthesized esters efficiently, and the synthetic efficiency even reached 81.5%. The physicochemical and catalytic properties of the lipase are extensively investigated for its potential industrial applications.     

Biochemical and hydrolytic properties of multiple thermostable α-galactosidases from Streptomyces griseoloalbus: Obvious existence of a novel galactose-tolerant enzyme

The multiple α-galactosidases from Streptomyces griseoloalbus—α-Gal I, α-Gal II and α-Gal III were purified to homogeneity by a two-step chromatographic process. The molecular masses and pI of the three enzymes were 72, 57 and 35 kDa, and 4.41, 5.6 and 6.13, respectively. α-Gal I showed N-terminal sequence homology to S. coelicolor A3(2) family 27 α-galactosidase. The optimum pH and temperature of the three α-galactosidases were 5.0, 6.5 and 5.5 and 65, 50 and 55 °C, respectively. α-Gal I was stable up to 65 °C and α-Gal II and α-Gal III up to 55 °C for 2 h. Based on the hydrolytic properties α-Gal I could be classified as a member of GH27 family and α-Gal II and α-Gal III as members of GH36 family. Metal cations like Hg²⁺, Ag²⁺ and Cu²⁺ inhibited enzyme activity while Mg²⁺ enhanced the activity of α-Gal I. Interestingly α-Gal I showed unusual tolerance to even higher concentrations of galactose, unlike the other two α-galactosidases, which were competitively inhibited by galactose. Melibiose was a competitive inhibitor of all three enzymes. Histidine, tryptophan and carboxylic residues were essential for catalytic action of the three α-galactosidases.     

Extracellular cold-active lipase of Microbacterium luteolum isolated from Gangotri glacier,western Himalaya: Isolation,partial purification and characterization

A psychrophilic bacterium producing cold-active lipase upon growth at low temperature was isolated from the soil samples of Gangotri glacier and identified as Microbacterium luteolum. The bacterial strain produced maximum lipase at 15 °C, at a pH of 8.0. Beef extract served as the best organic nitrogen source and ammonium nitrate as inorganic for maximum lipase production. Castor oil served as an inducer and glucose served as an additional carbon source for production of cold-active lipase. Ferric chloride as additional mineral salt in the medium, highly influenced the lipase production with an activity of 8.01 U ml^?1. The cold-active lipase was purified to 35.64-fold by DEAE-cellulose column chromatography. It showed maximum activity at 5 °C and thermostability up to 35 °C. The purified lipase was stable between pH 5 and 9 and the optimal pH for enzymatic hydrolysis was 8.0. Lipase activity was stimulated in presence of all the solvents (5%) tested except with acetonitrile. Lipase activity was inhibited in presence of Mn²⁺, Cu²⁺, and Hg²⁺; whereas Fe⁺, Na⁺ did not have any inhibitory effect on the enzyme activity. The purified lipase was stable in the presence of SDS; however, EDTA and dithiothreitol inhibited enzyme activity. Presence of Ca²⁺ along with inhibitors stabilized lipase activity. The cold active lipase thus exhibiting activity and stability at a low temperature and alkaline pH appears to be practically useful in industrial applications especially in detergent formulations.     

C-Terminal proline-rich sequence broadens the optimal temperature and pH ranges of recombinant xylanase from Geobacillus thermodenitrificans C5

Efficient utilization of hemicellulose entails high catalytic capacity containing xylanases. In this study, proline rich sequence was fused together with a C-terminal of xylanase gene from Geobacillus thermodenitrificans C5 and designated as GthC5ProXyl. Both GthC5Xyl and GthC5ProXyl were expressed in Escherichia coli BL21 host in order to determine effect of this modification. The C-terminal oligopeptide had noteworthy effects and instantaneously extended the optimal temperature and pH ranges and progressed the specific activity of GthC5Xyl. Compared with GthC5Xyl, GthC5ProXyl revealed improved specific activity, a higher temperature (70 °C versus 60 °C) and pH (8 versus 6) optimum, with broad ranges of temperature and pH (60–80 °C and 6.0–9.0 versus 40–60 °C and 5.0–8.0, respectively). The modified enzyme retained more than 80% activity after incubating in xylan for 3 h at 80 °C as compared to wild −type with only 45% residual activity. Our study demonstrated that proper introduction of proline residues on C-terminal surface of xylanase family might be very effective in improvement of enzyme thermostability. Moreover, this study reveals an engineering strategy to improve the catalytic performance of enzymes.     

Construction of a fusion enzyme of dextransucrase and dextranase: Application for one-step synthesis of isomalto-oligosaccharides

The linear isomalto-oligosaccharides (IMO) with DP2–DP10 were produced by one-step process using engineered fusion enzyme (DXSR) of endo-dextranase and only α-(1–6) glucan synthesizing dextransucrase. The fusion enzyme was successfully expressed in Escherichia coli and characterized. Compared to individual enzymes, DXSR had 150% increased endo-dextranase activity and 98% decreased dextransucrase activity. The partially purified DXSR displayed molecular mass of 240 kDa as analyzed by SDS–PAGE. It showed both enzyme activities on analysis by zymogram. The thermal- and pH-stability of DXSR was around 28 °C and pH at 5.0–6.4, respectively. IMOs production by DXSR was increased by the addition of metal ions such as Fe²⁺, Li⁺, K⁺ and Ni²⁺, but the enzyme was strongly inhibited by Hg²⁺ and Ag⁺. DXSR produced linear IMO with DP2–DP10 using sucrose as a sole substrate. The molecular weight and amount of IMO could be controlled by the sucrose concentration. DXSR gave 30-fold higher production of IMO than that of an equal activity mixture of the two enzymes such as dextranase and dextransucrase.     

Arginase from Bacillus thuringiensis SK 20.001: Purification,characteristics, and implications for l-ornithine biosynthesis

An l-ornithine high producing strain Bacillus thuringiensis SK20.001 was screened by our laboratory. An intracellular arginase used to biosynthesize l-ornithine from the strain was purified and characterized. The final specific arginase activity was 589.2 units/mg, with 70.1 fold enrichment and 22.4% recovery. The molecular weight of the enzyme was approximately 33,000 Da as evaluated by SDS-PAGE and 191,000 Da as determined by gel filtration. The enzyme had an optimum pH of 10.0 and an optimum temperature of 40 °C. It was stable from pH 8.0–12.0 and <50 °C without Mn²⁺. The presence of Mn²⁺ and Ni²⁺ had strong effects on the enzyme activity, and Mn²⁺ significantly increased the thermal stability of the enzyme. The arginase was slightly inhibited by Ca²⁺, Fe²⁺ and Zn²⁺. Trp, Asp, Glu, Tyr, and Arg residues were directly involved in the arginase activity evaluated by chemical modifications. The K_m and V_max for l-arginine were estimated to be 15.6 mM and 538.9 μmol/min/mg. The biosynthesis yield of l-ornithine was 72.7 g/L with the enzyme.     

Effects of galactomannan as carbon source on production of α-galactosidase by Thermomyces lanuginosus: Fermentation,purification and partial characterisation

Thermophilic fungus Thermomyces lanuginosus CBS 395.62/b strain is able to grow and synthesise extracellular α-galactosidase in media containing galactomannan such as locust bean gum (LBG) or guar gum (GG). Production of extracellular α-galactosidase was enhanced from 1.2 U/mL to 4–6 U/mL meaning about 3–5 times increase by optimisation of medium composition. This enzyme was purified to homogeneity by partial precipitation with 2-propanol and different liquid chromatographical steps. The developed purification protocol yielded 22% of enzyme activity with 900 purified fold. Molecular mass of the purified α-galactosidase enzyme was estimated to be 53 kDa. Maximal catalytic activity of the enzyme was obtained in the acidic pH range between pH 4.6 and 4.8 and in the temperature range 60–66 °C. More than 95% of enzyme activity was remaining after 1-day incubation at 70 °C and on pH in the range from 4.0 to 7.0. The enzyme activity was significantly stimulated by Mg²⁺, Mn²⁺ and K⁺ ions, while considerably inhibited by the presence of Ca²⁺, Ag⁺ and Hg²⁺.     

Novel antioxidant bromophenols with acetylcholinesterase,butyrylcholinesterase and carbonic anhydrase inhibitory actions

In this study, a series of novel bromophenols were synthesized from benzoic acids and methoxylated bromophenols. The synthesized compounds were evaluated by using different bioanalytical antioxidant assays including 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS⁺) radical scavenging assays. Also, reducing power of novel bromophenols were evaluated by Cu²⁺-Cu⁺ reducing, Fe³⁺-Fe²⁺ reducing and [Fe³⁺-(TPTZ)₂]³⁺-[Fe²⁺-(TPTZ)₂]₂+ reducing and ferrous ions (Fe²⁺) chelating abilities. The compounds demonstrate powerful antioxidant activities when compared to standard antioxidant molecules of α-tocopherol, trolox, butylated hydroxyanisole (BHA), and butylated hydroxytoluene (BHT). Also in the last part of this studies novel bromophenols were tested against some metabolic enzymes including acetylcholinesterase (AChE), butyrylcholinesterase (BChE) enzymes and carbonic anhydrase I, and II (hCA I and hCA II) isoenzymes. The newly synthesized bromophenols showed Ki values in a range of 6.78 ± 0.68 to 126.07 ± 35.6 nM against hCA I, 4.32 ± 0.23 to 72.25 ± 12.94 nM against hCA II, 4.60 ± 1.15 to 38.13 ± 5.91 nM against AChE and 7.36 ± 1.31 to 29.38 ± 3.68 nM against BChE.     

Molecular cloning and characterization of a novel thermostable xylanase from Paenibacillus campinasensis BL11

An open reading frame (XylX) with 1131 nucleotides from Paenibacillus campinasensis BL11 was cloned and expressed in E. coli. It encodes a family 11 endoxylanase, designated as XylX, of 41 kDa. The homology of the amino acid sequence deduced from XylX is only 73% identical to the next closest sequence. XylX contains a family 11 catalytic domain of the glycoside hydrolase and a family 6 cellulose-binding module. The recombinant xylanase was fused to a His-tag for affinity purification. The XylX activity was 2392 IU/mg, with a K_m of 6.78 mg/ml and a V_max of 4953 mol/min/mg under optimal conditions (pH 7, 60 °C). At pH 11, 60 °C, the activity was still as high as 517 IU/mg. Xylanase activities at 60 °C under pH 5 to pH 9 remained at more than 69.4% of the initial activity level for 8 h. The addition of Hg²⁺ at 5 mM almost completely inhibited xylanase activity, whereas the addition of tris-(2-carboxyethyl)-phosphine (TCEP) and 2-mercaptoethanol stimulated xylanase activity. No relative activities for Avicel, CMC and d-(+)-cellobiose were found. Xylotriose constitutes the majority of the hydrolyzed products from oat spelt and birchwood xylan. Broad pH and temperature stability shows its application potentials for biomass conversion, food and pulp/paper industries.