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.

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.

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.

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.