Biochemical characterization of an alkaline and detergent-stable Lipase from Fusarium annulatum Bugnicourt strain CBS associated with olive tree dieback.

This work describes a novel extracellular lipolytic carboxylester hydrolase named FAL, with lipase and phospholipase A1 (PLA1) activity, from a newly isolated filamentous fungus Ascomycota CBS strain, identified as Fusarium annulatum Bunigcourt. FAL was purified to about 62-fold using ammonium sulphate precipitation, Superdex® 200 Increase gel filtration and Q-Sepharose Fast Flow columns, with a total yield of 21%. The specific activity of FAL was found to be 3500 U/mg at pH 9 and 40°C and 5000 U/mg at pH 11 and 45°C, on emulsions of triocanoin and egg yolk phosphatidylcholine, respectively. SDS-PAGE and zymography analysis estimated the molecular weight of FAL to be 33 kDa. FAL was shown to be a PLA1 with a regioselectivity to the sn-1 position of surface-coated phospholipids esterified with α-eleostearic acid. FAL is a serine enzyme since its activity on triglycerides and phospholipids was completely inhibited by the lipase inhibitor Orlistat (40 µM). Interestingly, compared to Fusarium graminearum lipase (GZEL) and the Thermomyces lanuginosus lipase (Lipolase®), this novel fungal (phospho)lipase showed extreme tolerance to the presence of non-polar organic solvents, non-ionic and anionic surfactants, and oxidants, in addition to significant compatibility and stability with some available laundry detergents. The analysis of washing performance showed that it has the capability to efficiently eliminate oil-stains. Overall, FAL could be an ideal choice for application in detergents.

Preparation, characterization, immobilization, and molecular docking analysis of a novel detergent-stable subtilisin-like serine protease from Streptomyces mutabilis strain TN-X30.

We recently described the production of a detergent-biocompatible crude protease from Streptomyces mutabilis strain TN-X30. Here, we describe the purification, characterization, and immobilization of the serine alkaline protease (named SPSM), as well as the cloning, sequencing, and over-expression of its corresponding gene (spSM). Pure enzyme was obtained after ammonium sulphate precipitation followed by heat-treatment and Sephacryl® S-200 column purification. The sequence of the first 26 NH2-terminal residues of SPSM showed a high sequence identity to subtilisin-like serine proteases produced by actinobacteria. The spSM gene was heterologously expressed in Escherichia coli BL21(DE3)pLysS and E. coli BL21-AI™ strains using pTrc99A (rSPSM) and Gateway™ pDEST™ 17 [(His)6-tagged SPSM] vectors, respectively. Results obtained indicated that the (His)6-tagged SPSM showed the highest stability. The SPSM was immobilized using encapsulation and adsorption-encapsulation approaches and three different carriers. Features of SPSM in soluble and immobilized forms were analyzed by Fourier transform infrared (FTIR) spectroscopy in attenuated total reflection (ATR) mode, X-ray diffraction (XRD), zeta potential measurements, and field emission scanning electron microscopy (FE-SEM). The white clay and kaolin used in this study are eco-friendly binders to alginate-SPSM and show great potential for application of the immobilized SPSM in various industries. Molecular modeling and docking of N-succinyl-l-Phe-l-Ala-l-Ala-l-Phe-p-nitroanilide in the active site of SPSM revealed the involvement of 21 amino acids in substrate binding.

Heterologous expression and purification of keratinase from Actinomadura viridilutea DZ50: feather biodegradation and animal hide dehairing bioprocesses.

The keratin-degrading bacterium Actinomadura viridilutea DZ50 secretes a keratinase (KERDZ) with potential industrial interest. Here, the kerDZ gene was extracellularly expressed in Escherichia coli BL21(DE3)pLysS using pTrc99A vector. The recombinant enzyme (rKERDZ) was purified and biochemically characterized. Results showed that the native and recombinant keratinases have similar biochemical characteristics. The conventional dehairing with lime and sodium sulfide degrades the hair to the extent that it cannot be recovered. Thus, these chemical processes become a major contributor to wastewater problem and create a lot of environmental concern. The complete dehairing was achieved with 2000 U/mL rKERDZ for 10 h at 40 °C. In fact, keratinase assisted dehairing entirely degraded chicken feather (45 mg) and removed wool/hair from rabbit, sheep, goat, or bovine' hides (1.6 kg) while preserving the collagen structure. The enzymatic process is the eco-friendly option that reduces biological (BOD) (50%) and chemical (COD) oxygen demands (60%) in leather processing. Consequently, the enzymatic hair removal process could solve the problem of post-treatments encountering the traditional leather processing. The enzymatic (rKERDZ) dehaired leather was analyzed by scanning electron microscopic (SEM) studies, which revealed similar fiber orientation and compactness compared with control sample. Those properties support that the rKERDZ enzyme-mediated process is greener to some extent than the traditional one.

Biochemical and molecular characterization of an acido-thermostable endo-chitinase from Bacillus altitudinis KA15 for industrial degradation of chitinous waste.

This paper reports the isolation and identification of an acido-thermostable chitinase (ChiA-Ba43) which was purified, from the culture liquid of Bacillus altitudinis strain KA15, and characterized. Purification of ChiA-Ba43 produced a 69.6-fold increase in the specific activity (120,000 U/mg) of the chitinase, with a yield of 51% using colloidal chitin as substrate. ChiA-Ba43 was found to be a monomeric protein with a molecular mass of 43,190.05 Da as determined by MALDI-TOF/MS. N-terminal sequence of the first 27 amino-acids (aa) of ChiA-Ba43 displayed homology to chitinases from other Bacillus species. Interestingly, ChiA-Ba43 exhibited optimum pH and temperature of 4-5.5 and 85 °C, respectively. Thin-layer chromatography (TLC) showed that the final hydrolyzed products of the enzyme from chitin-oligosaccharides and colloidal chitin are a mixture of (GlcNAc)2, (GlcNAc)3, (GlcNAc)4, and (GlcNAc)5, which indicates that ChiA-Ba43 possesses an endo-acting function. More interestingly, compared to ChiA-Mt45, ChiA-Hh59, Chitodextrinase®, N-acetyl-ß-glucosaminidase®, and ChiA-65, ChiA-Ba43 demonstrated a high level of catalytic efficiency and outstanding tolerance towards various organic solvents. The chiA-Ba43 gene (1332 bp) encoding ChiA-Ba43 (409 aa) was cloned, sequenced, and expressed in Escherichia coli strain HB101. The biochemical properties of the recombinant chitinase (rChiA-Ba43) were equivalent to those of the natively expressed enzyme. These properties make ChiA-Ba43 an ideal candidate for industrial bioconversion of chitinous waste.

Identification and homology modeling of a new biotechnologically compatible serine alkaline protease from moderately halotolerant Gracilibacillus boraciitolerans strain LO15.

A new serine alkaline protease (designated as SAPGB) from Gracilibacillus boraciitolerans strain LO15, was produced (9000 U/mL), purified, and characterized. SAPGB has a monomer structure with a precise molecular weight of 30,285.03 kDa as learnt from matrix-assisted laser desorption/ionization-time of flight/mass spectroscopy (MALDI-TOF/MS) exploration. The NH2-terminal amino-acid succession revealed significant identity with Bacillus proteases. The SAPGB was irreversibly inhibited by diiodopropyl fluorophosphates (DFP) and phenylmethylsulfonyl fluoride (PMSF). The enzyme displayed optimum activity at 65 °C and pH 10. The maximal activity was achieved in the range 0.5-5 M NaCl and about 52% of the activity was preserved across the broad salinity range of 0-30%. SAPGB exhibited a considerable catalytic efficiency (ratio kcat/Km) and degree of hydrolysis (DH). In addition, SAPGB showed a high tolerance to several organic solvents and an excellent detergent compatibility than SAPV, SAPA, Thermolysin type X, and Esperase 8.0 L. These properties make SAPGB a potential candidate for detergent formulations. On the other hand, sapGB gene was cloned and expressed in E. coli BL21(DE3)pLysS and the biochemical properties of the purified extracellular recombinant protease (rSAPGB) were similar to those of SAPGB. Finally, a 3D structural model of SAPGB was constructed by homology modeling.

A thermophilic and thermostable xylanase from Caldicoprobacter algeriensis: Recombinant expression, characterization and application in paper biobleaching.

A novel xylanase gene xynBCA, encoding a polypeptide of 439 residues (XynBCA), was cloned from Caldicoprobacter algeriensis genome and recombinantly expressed in Escherichia coli BL21(DE3). The amino acid sequence analysis showed that XynBCA belongs to the glycoside hydrolase family 10. The purified recombinant enzyme has a monomeric structure of 52 kDa. It is active and stable in a wide range of pH from 3 to 10 with a maximum activity at 6.5. Interestingly, XynBCA was highly thermoactive with an optimum temperature of 80 °C, thermostable with a half-life of 20 min at 80 °C. The specific activity was 117 U mg-1, while the Km and Vmax were 1.247 mg ml-1, and 114.7 µmol min-1 mg-1, respectively. The investigation of XynBCA in kraft pulp biobleaching experiments showed effectiveness in releasing reducing sugars and chromophores, with best achievements at 100 U g-1 of pulp and 1 h of incubation. The comparative molecular modeling studies with the less thermostable Xylanase B from Clostridium stercorarium, revealed extra charged residues at the surface of XynBCA potentially participating in the formation of intermolecular hydrogen bonds with solvent molecules or generating salt bridges, therefore contributing to the higher thermal stability.

α-Amylase production by Tepidimonas fonticaldi strain HB23: statistical optimization and compatibility study for use in detergent formulations.

In a previous study, a thermostable α-amylase-producing bacterium (designated HB23) was isolated from an Algerian hydrothermal spring. In the present study, the native strain was subjected to a statistical optimization aimed at enhancing the α-amylase production. To achieve this, thirteen factors have been studied, among which are cultural and nutritional parameters. Wheat bran, a by-product of the grain milling industry, was the factor that positively influenced α-amylase production. A modified L27 Taguchi design was used to screen these factors. Furthermore, a Box-Behnken matrix, supplemented by the use of response surface methodology (RSM), allowed for the identification of optimum levels of the following factors: a 1% inoculum size, 15 g/L soluble starch, 5 g/L wheat bran, and 1 g/L tryptone. Optimized conditions resulted in an amylolytic activity of 320 U/mL, which is a tenfold increase when compared with unoptimized production level. Phenotypical and molecular identification of strain HB23 revealed its close relationship to various Tepidimonas strains, specifically to Tepidimonas fonticaldi. The crude enzyme preparation turned out to be compatible with various laundry detergents and led to a substantial improvement in their washing performance. A comparison of the performance of the crude enzyme preparation with that of the commercial α-amylase (Termamyl® 300 L) highlights the potential of the HB23 enzyme as a bio-additive in detergent formulations.

A biological clean processing approach for the valorization of speckled shrimp Metapenaeus monoceros by-product as a source of bioactive compounds.

The efficiency of the proteolytic strain Anoxybacillus kamchatkensis M1V in the fermentation of speckled shrimp by-product was investigated for the recovery of a deproteinized bioactive hydrolysate. The biological activities of the resulting hydrolysate were also examined by applying several antioxidant and enzyme inhibitory assays. The strain M1V was found to produce high level of protease activity (2000 U/mL) when grown in media containing only shrimp powder at 25 g/L. The crude protease displayed a significant deproteinization capabiliy, with the best efficiency (48%) being recorded for an enzyme to substrate (E/S) ratio of 30 U/mg. Following the deproteinization, chitin was recovered and the authenticity was confirmed by Fourier-transform infrared spectroscopy (FTIR) analysis. On the other hand, the obtained hydrolysate showed a significant enzymatic inhibitory potential against acetylcholinesterase, tyrosinase, amylase, and angiotensin I convertase, and a strong antioxidant activity. Graphical Abstract.

Identification of a New Serine Alkaline Peptidase from the Moderately Halophilic Virgibacillus natechei sp. nov., Strain FarDT and its Application as Bioadditive for Peptide Synthesis and Laundry Detergent Formulations.

A new peptidase designated as SAPV produced from a moderately halophilic Virgibacillus natechei sp. nov., strain FarDT was investigated by purification to homogeneity followed by biochemical and molecular characterization purposes. Through optimization, it was determined that the optimum peptidase activity was 16,000 U/mL. It was achieved after 36 h incubation at 35°C in the optimized enzyme liquid medium (ELM) at pH 7.4 that contains only white shrimp shell by-product (60 g/L) as sole energy and carbon sources. The SAPV enzyme is a monomer protein with a molecular mass of 31 kDa as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and high-performance liquid chromatography (HPLC) gel filtration chromatography. The sequence of its NH2-terminal amino-acid residues showed homology with those of Bacillus peptidases S8/S53 superfamily. The SAPV showed optimal activity at pH 9 and 60°C. Irreversible inhibition of enzyme activity by diiodopropyl fluorophosphates (DFP) and phenylmethanesulfonyl fluoride (PMSF) confirmed its belonging to the serine peptidases. Considering its interesting biochemical characterization, the sapV gene was cloned, sequenced, and heterologously overexpressed in the extracellular fraction of E. coli BL21(DE3)pLysS. The biochemical properties of the recombinant peptidase (rSAPV) were similar to those of the native one. The highest sequence identity value (97.66%) of SAPV was obtained with peptidase S8 from Virgibacillus massiliensis DSM 28587, with 9 amino-acid residues of difference. Interestingly, rSAPV showed an outstanding and high resistance to several organic solvents than SPVP from Aeribacillus pallidus VP3 and Thermolysin type X. Furthermore, rSAPV exhibited an excellent detergent stability and compatibility than Alcalase 2.4 L FG and Bioprotease N100L. Considering all these remarkable properties, rSAPV has attracted the interest of industrialists.

Identification of a novel protease from the thermophilic Anoxybacillus kamchatkensis M1V and its application as laundry detergent additive.

A thermostable extracellular alkaline protease (called SAPA) was produced (4600 U/mL) by Anoxybacillus kamchatkensis M1V, purified to homogeneity, and biochemically characterized. SAPA is a monomer with a molecular mass of 28 kDa estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Native-PAGE, casein-zymography, and size exclusion using high performance liquid chromatography (HPLC). The sequence of its NH2-terminal amino-acid residues showed high homology with those of Bacillus proteases. The SAPA irreversible inhibition by diiodopropyl fluorophosphates (DFP) and phenylmethanesulfonyl fluoride (PMSF) confirmed its belonging to the serine proteases family. Optimal activity of SAPA was at pH 11 and 70 °C. The sapA gene was cloned and expressed in the extracellular fraction of E. coli. The highest sequence identity value (95%) of SAPA was obtained with peptidase S8 from Bacillus subtilis WT 168, but with 16 amino-acids of difference. The biochemical characteristics of the purified recombinant extracellular enzyme (called rSAPA) were analogous to those of native SAPA. Interestingly, rSAPA exhibit a degree of hydrolysis that were 1.24 and 2.6 than SAPB from Bacillus pumilus CBS and subtilisin A from Bacillus licheniformis, respectively. Furthermore, rSAPA showed a high detergent compatibility and an outstanding stain removal capacity compared to commercial enzymes: savinase™ 16L, type EX and alcalase™ Ultra 2.5 L.

Purification and biochemical characterization of a new organic solvent-tolerant chitinase from Paenibacillus timonensis strain LK-DZ15 isolated from the Djurdjura Mountains in Kabylia, Algeria.

A new extracellular chitinase (called ChiA-Pt70) was produced and purified from a newly isolated Paenibacillus timonensis strain LK-DZ15. The maximum chitinase activity recorded after 44-h of incubation at 30 °C was 11,500 U/mL. Pure enzyme was obtained after ammonium sulphate precipitation (40-70%) followed by sequential column chromatographies on fast performance liquid chromatography (FPLC) and high performance liquid chromatography (HPLC). Based on matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis, the purified enzyme is a monomer with a molecular mass of 70,166.11 kDa. The sequence of the 25 NH2-terminal residues of the mature ChiA-70 showed high homology with Paenibacillus GH-18 chitinases family. Optimal activity was achieved at pH 4.5 and 80 °C. The pure enzyme was completely inhibited by p-chloromercuribenzoic acid (p-CMB), 5,5'-dithio-bis-2-nitro benzoic acid (DTNB), and N-ethylmaleimide (NEM). Chitinase activity was high on colloidal chitin, chitin azure, glycol chitin, glycol chitosane, chitotriose, and chito-oligosaccharide while it did not hydrolyse chitibiose and amylose. Furthermore, thin-layer chromatography (TLC) analysis from enzymatic catalyzed hydrolysis of colloidal chitin showed that ChiA-Pt70 acted as an endo-splitting enzyme. Its Km and kcat values were 0.611 mg colloidal chitin/mL and 87,800 s-1, respectively. Interestingly, its catalytic efficiency was higher than those of chitinases ChiA-Mt45 from Melghiribacillus thermohalophilus strain Nari2AT, ChiA-Hh59 from Hydrogenophilus hirchii strain KB-DZ44, Chitodextrinase® from Streptomyces griseus, and N-acetyl-ß-glucosaminidase® from Trichoderma viride. Therefore, ChiA-Pt70 exhibited remarkable biochemical properties suggesting that it is suitable for the enzymatic degradation of chitin.

Purification and biochemical characterization of a novel thermostable and halotolerant subtilisin SAPN, a serine protease from Melghiribacillus thermohalophilus Nari2AT for chitin extraction from crab and shrimp shell by-products.

The present study investigates the purification and biochemical characterization of a novel extracellular serine alkaline protease, subtilisin (called SAPN) from Melghiribacillus thermohalophilus Nari2AT. The highest yield of protease (395 IU/g) with white shrimp shell by-product (40 g/L) as a unique source of nutriments in the growth medium was achieved after 52 h at 55 °C. The monomeric enzyme of about 30 kDa was purified to homogeneity by ammonium sulfate fractionation, heat treatment, followed by sequential column chromatographies. The optimum pH and temperature values for subtilisin activity were pH 10 and 75 °C, respectively, and half lives of 9 and 5 h at 80 and 90 °C, respectively. The sequence of the 25 NH2-terminal residues pertaining of SAPN exhibited a high homology with those of Bacillus subtilisins. The inhibition by DFP and PMSF indicates that this enzyme belongs to the serine proteases family. SAPN was found to be effective in the deproteinization (DDP %) of blue swimming crab (Portunus segnis) and white shrimp (Metapenaeus monoceros) by-products, with a degree of 65 and 82%, respectively. The commercial and the two chitins obtained in this work showed a similar peak pattern in Fourier-Transform Infrared (FTIR) analysis, suggesting that SAPN is suitable for the bio-production of chitin from shell by-products.

Purification, biochemical, and molecular characterization of a novel extracellular thermostable and alkaline α-amylase from Tepidimonas fonticaldi strain HB23.

The present study investigated the purification, biochemical, and molecular characterization of a novel thermostable α-amylase (TfAmy48) from Tepidimonas fonticaldi strain HB23. MALDI-TOF/MS analysis indicated that the purified enzyme is a monomer with a molecular mass of 48,138.10 Da. The results from amino-acid sequence analysis revealed high homology between the 25 NH2-terminal residues of TfAmy48 and those of Gammaproteobacteria α-amylases. The optimum pH and temperature values for α-amylase activity were pH 8 and 80 °C, respectively. Thin-layer chromatography (TLC) analysis showed that the final hydrolyzed products of the enzyme from soluble potato starch were maltopentaose, maltose, and maltotriose, which indicate that TfAmy48 possessed an endo-acting pattern. Compared to Termamyl®300 L, TfAmy48 showed extreme stability and tolerance towards organic solvents and excellent compatibility with some commercial laundry detergents. These proprieties make TfAmy48 enzyme a potential candidate as a cleaning bioadditive in detergent composition. The Tfamy48 gene encoding TfAmy48 was cloned, sequenced, and heterologously-expressed in the extracellular fraction of Escherichia coli strain BL21(DE3)pLysS. The biochemical properties of the extracellular purified recombinant enzyme (rTfAmy48) were similar to those of native one. The highest sequence identity value (97%) was obtained with PsAmy1 α-amylase from Pseudomonas sp. strain KFCC10818, with only 16 amino-acid (aa) residues of difference.

A novel thermostable and efficient Class II glucose isomerase from the thermophilic Caldicoprobacter algeriensis: Biochemical characterization, molecular investigation, and application in High Fructose Syrup production.

A novel glucose isomerase gene from the thermophilic Caldicoprobacter algeriensis, encoding a polypeptide of 438 residues, was identified, cloned and successfully expressed in E. coli. The purified enzyme (GICA) was a homotetramer of about 200 kDa displaying the highest activity at pH 7.0 and 90 °C and retaining 97% of its maximum activity at pH 6.5. The enzyme showed an excellent thermostability with a half-life of 6 min at 100 °C. Interestingly, GICA had a very high affinity of 40 mM and catalytic efficiency of 194 min-1 mM-1 toward d-glucose at 90 °C. A maximum of 54.7% d-glucose to d-fructose conversion was achieved by GICA at 85 °C making it an attractive candidate for HFCS-55 production. The primary sequence inspection and molecular modeling studies revealed that the thermal stability of GICA could be attributed to the presence of extra charged residues at the surface like E108 and Q408 increasing surface charge interactions. Moreover, a serine at position 56 near to P58 could establish hydrogen bond strengthening the dimer attachment. The high catalytic efficiency and affinity of GICA could be ascribed to the presence of amino acid like E108 and K62 that created more charges around the catalytic site entry.

Purification and biochemical characterization of a novel acido-halotolerant and thermostable endochitinase from Melghiribacillus thermohalophilus strain Nari2AT.

An extracellular acido-thermostable endochitinase (called ChiA-Mt45) from thermohalophilic Melghiribacillus thermohalophilus strain Nari2AT gen. nov. sp. nov., was purified and biochemically characterized. The maximum chitinase activity recorded after 48-h of incubation at 55 °C was 9000 U/mL. Pure enzyme was obtained after heat treatment (20 min at 90 °C) followed by sequential column chromatographies on fast performance liquid chromatography (FPLC) and high performance liquid chromatography (HPLC). Based on MALDI-TOF/MS analysis, the purified enzyme is a monomer with a molecular mass of 45201.10 Da. The 27 residue NH2-terminal sequence of the enzyme showed high homology with Bacillus GH-18 chitinases family. The optimum pH and temperature values for chitinase activity were pH 3.5 and 90 °C, respectively. In addition, the enzyme was halotolerant and can be classified as an extremozyme. The pure enzyme was completely inhibited by p-chloromercuribenzoic acid (p-CMB) and N-ethylmaleimide (NEM). Its Km and kcat values were 0.253 mg colloidal chitin/mL and 47000 s-1, respectively. Interestingly, its catalytic efficiency was higher than those of chitinases ChiA-Hh59 from Hydrogenophilus hirchii KB-DZ44 and chitodextrinase from Streptomyces griseus, and N-acetyl-ß-glucosaminidase from Trichoderma viride. The studied chitinase exhibited high activity towards colloidal chitin, chitin azure, glycol chitin, while it did not hydrolyse chitibiose and amylose. Additionally, thin-layer chromatography (TLC) analysis from chitin-oligosaccharides showed that ChiA-Mt45 acted as an endosplitting enzyme. Overall, the chitinase ChiA-Mt45 may have great potential for the enzymatic degradation of chitin.

Physical and enzymatic properties of a new manganese peroxidase from the white-rot fungus Trametes pubescens strain i8 for lignin biodegradation and textile-dyes biodecolorization.

A new manganese peroxidase-producing white-rot basidiomycete fungus was isolated from symptomatic wood of the camphor trees Cinnamomum camphora (L.) at the Hamma Botanical Garden (Algeria) and identified as Trametes pubescens strain i8. The enzyme was purified (MnP TP55) to apparent electrophoretic homogeneity and biochemically characterized. The specific activity and Reinheitzahl value of the purified enzyme were 221 U/mg and 2.25, respectively. MALDI-TOF/MS analysis revealed that the purified enzyme was a monomer with a molecular mass of 55.2 kDa. The NH2-terminal sequence of the first 26 amino acid residues of MnP TP55 showed high similarity with those of white-rot fungal peroxidases. It revealed optimal activity at pH 5 and 40 °C. This peroxidase was completely inhibited by sodium azide and potassium cyanide, suggesting the presence of heme-components in its tertiary structure. Interestingly, MnP TP55 showed higher catalytic efficiency, organic solvent-tolerance, dye-decolorization ability, and detergent-compatibility than that of horseradish peroxidase (HRP) from roots of Armoracia rustanica, manganese peroxidase from Bjerkandera adusta strain CX-9 (MnP BA30), and manganese peroxidase from Phanerochaete chrysosporium (MnP PC). Overall, the findings provide strong support for the potential candidacy of MnP TP55 for environmental applications, mainly the development of enzyme-based technologies for lignin biodegradation, textile-dyes biodecolorization, and detergent formulations.

Purification and characterization of two novel peroxidases from the dye-decolorizing fungus Bjerkandera adusta strain CX-9.

Two extracellular peroxidases from Bjerkandera adusta strain CX-9, namely a lignin peroxidase (called LiP BA45) and manganese peroxidase (called MnP BA30), were purified simultaneously by applying successively, ammonium sulfate precipitation-dialysis, Mono-S Sepharose anion-exchange and Sephacryl S-200 gel filtration and biochemically characterized. The sequence of their NH2-terminal amino acid residues showed high homology with those of fungi peroxidases. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis revealed that the purified enzymes MnP BA30 and LiP BA45 were a monomers with a molecular masses 30125.16 and 45221.10Da, respectively. While MnP BA30 was optimally active at pH 3 and 70°C, LiP BA45 showed optimum activity at pH 4 and 50°C. The two enzymes were inhibited by sodium azide and potassium cyanide, suggesting the presence of heme-components in their tertiary structures. The Km and Vmax for LiP BA45 toward 2,4-Dichlorolphenol (2,4-DCP) were 0.099mM and 9.12U/mg, respectively and for MnP BA30 toward 2,6-Dimethylphenol (2,6-DMP), they were 0.151mM and 18.60U/mg, respectively. Interestingly, MnP BA30 and LiP BA45 demonstrated higher catalytic efficiency than that of other tested peroxidases (MnP, LiP, HaP4, and LiP-SN) and marked organic solvent-stability and dye-decolorization efficiency. Data suggest that these peroxidases may be considered as potential candidates for future applications in distaining synthetic-dyes.

Biochemical characterization of a novel thermostable chitinase from Hydrogenophilus hirschii strain KB-DZ44.

An extracellular acido-thermostable endo-chitinase (called ChiA-Hh59) from thermophilic Hydrogenophilus hirschii strain KB-DZ44, was purified and characterized. The maximum chitinase activity recorded after 36-h of incubation at 60°C was 3000U/ml. Pure enzyme was obtained after heat and acidic treatment, precipitation by ammonium sulphate and acetone, respectively, followed by sequential column chromatographies on Sephacryl S-200 and Mono Q-Sepharose. Based on Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis, the purified enzyme is a monomer with a molecular mass of 59103.12-Da. The 22 residue NH2-terminal sequence of the enzyme showed high homology with family-18 bacterial chitinases. The optimum pH and temperature values for chitinase activity were pH 5.0 and 85°C, respectively. The pure enzyme was completely inhibited by p-chloromercuribenzoic acid (p-CMB) and N-ethylmaleimide (NEM). The obtained results suggest that ChiA-Hh59 might be an endo-chitinase. The studied chitinase exhibited high activity towards colloidal chitin, chitin azure, glycol chitin, while it did not hydrolyse chitibiose and amylose. Its Km and kcat values were 0.298mg colloidal chitin/ml and 14400s-1, respectively. Its catalytic efficiency was higher than those of chitodextrinase and ChiA-65. Additionally, Thin-layer chromatography (TLC) analysis from chitin-oligosaccharides showed that ChiA-Hh59 acted as an endo-splitting enzyme. In conclusion, this chitinase may have great potential for the enzymatic degradation of chitin.

Biochemical and molecular characterization of a novel metalloprotease from Pseudomonas fluorescens strain TBS09.

A novel extracellular protease called MPDZ was purified and characterized from Pseudomonas fluorescens strain TBS09. The enzymatic properties of MPDZ were investigated using biochemical and biophysical methods. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis revealed that it was a monomer with a molecular mass of 50013.17Da. The NH2-terminal 27 amino acid sequence of MPDZ showed high homology with those of Pseudomonas-proteases of the serralysin family. MPDZ showed optimal activity at pH 7 and 60°C. It was totally inhibited by EGTA, EDTA, and 1,10-phenanthroline, suggesting its belonging to the metalloprotease family. Because of the interesting properties, the mpDZ gene encoding MPDZ was cloned, sequenced, and expressed in E. coli. The deduced amino acid sequence showed a strong homology with other Pseudomonas-metalloproteases. The highest sequence identity value (97%) was obtained with AprX from P. fluorescens strain CY091, with only 12 different amino acid residues. The physico-chemical properties of the extracellular purified recombinant enzyme (rMPDZ) were similar to those of MPDZ. Overall, MPDZ is bestowed with a number of promising biochemical properties that might give new opportunities for its biocatalytic applications. These data constitute an essential first step towards an understanding of the properties of MPDZ enzyme.

Biochemical properties of a new thermo- and solvent-stable xylanase recovered using three phase partitioning from the extract of Bacillus oceanisediminis strain SJ3.

The present study investigates the production and partial biochemical characterization of an extracellular thermostable xylanase from the Bacillus oceanisediminis strain SJ3 newly recovered from Algerian soil using three phase partitioning (TPP). The maximum xylanase activity recorded after 2 days of incubation at 37 °C was 20.24 U/ml in the presence of oat spelt xylan. The results indicated that the enzyme recovered in the middle phase of TPP system using the optimum parameters were determined as 50% ammonium sulfate saturation with 1.0:1.5 ratio of crude extract: t-butanol at pH and temperature of 8.0 and 10 °C, respectively. The xylanase was recovered with 3.48 purification fold and 107% activity recovery. The enzyme was optimally active at pH 7.0 and was stable over a broad pH range of 5.0-10. The optimum temperature for xylanase activity was 55 °C and the half-life time at this temperature was of 6 h. At this time point the enzyme retained 50% of its activity after incubation for 2 h at 95 °C. The crude enzyme resist to sodium dodecyl sulfate and ß-mercaptoethanol, while all the tested ions do not affect the activity of the enzyme. The recovered enzyme is, at least, stable in tested organic solvents except in propanol where a reduction of 46.5% was observed. Further, the stability of the xylanase was higher in hydrophobic solvents where a maximum stability was observed with cyclohexane. These properties make this enzyme to be highly thermostable and may be suggested as a potential candidate for application in some industrial processes. To the best of our knowledge, this is the first report of xylanase activity and recoverey using three phase partitioning from B. oceanisediminis.