Production and Characterisation of Thermostable Protease from Bacillus Stearothermophilus Strain F1

Screening and isolation of thermophilic proteolytic bacteria were carried out from composting areas, high temperature ponding systems and areas surrounding palm oil mills. Twelve isolates were positive on Skim Milk A gar (10%) of which 11 produced protease in culture broth. A thermophilic and highly proteolytic isolate identifi edas Bacillus stearothermophilus strain F1 isolated from decomposed oil palm branch, was selected for further study. B. stearothermophilus strain F1 could grow up to 80°C within a broad pH ranges (pH 5 to 11) with an optimal growth temperature and pH at 70°C and 9.5, respectively. The doubling time of this bacteria at 60°C was 70 min. Maximum protease production was achieved after 24 h cultivation when grown in 50 ml medium (pH 10.0) under shaking condition at 60°C. Static condition inhibited protease production but not growth. Cultures grown on peptone (iv) generated the highest amount of protease and lower production was observed when glucose or other rapidly metabolized carbon sources were added. Ammonium salt (0.5%) and amino acids (0.5%) interfered with protease formation whenever they were added to the medium. Although the protease production was calcium independ ent, the presence of the ion at 4.5 mM enhanced the yield by two-fold. Protease production occurred during the midexponential growth phase of the bacterial growth and its onset coincided with sporulation

Microbial enzymes: from earth to space

Biotechnology can provide an unlimited and pure source of enzymes as an alternative to the harsh chemicals traditionally used in industry for accelerating chemical reactions. Enzymes are found in naturally occurring microorganisms, such as bacteria, fungi and yeast, all of which may or may not be genetically modified. Hydrolytic enzymes, such as lipases and proteases are much sought after as the biocatalysts of the future. Proteases have been widely used in industry and there is always scope for new enzymes to be utilized in existing applications as well as new ones. Lipases on the other hand, are projected to have exciting potential in the advancement of the bioprocessing industry in particular oleochemicals. Thermostable enzymes are always sought by the industries while solvent tolerant enzymes are becoming the vogue in view of their ability to function in low aqueous medium, suitable for synthetic reactions. Advances in science and technology have allowed researchers to improve enzymes either through modifications of enzyme producing microorganisms, or via direct changes to the enzymes themselves. By studying the relations between the structure of a protein and how it functions, methods to improve and engineer enzymes can be developed. One of the most widely used methods in studying protein structures is crystallography which can provide an insight into the protein structures and functions from global folds to the atomic details of bonding. The crystals are analyzed by x-ray diffraction to determine their structures, but this procedure is only possible for large and relatively pure proteins. As protein crystals are fragile, it is difficult for some proteins to grow adequately large or to obtain perfect protein crystals in Earth-based laboratories. The influence of gravity on Earth distorts the shape of the crystals resulting in imperfections in the structures. Microgravity can provide an ideal environment for the growth of crystals. This is due to the fact that in a microgravity environment of space there are no gravity-induced effects such as sedimentation and convection that can disrupt the growth of these fragile protein crystals; thereby increasing the probability of growing larger, more perfect crystals. Crystals grown in microgravity generally have improved morphology, larger volume, better optical properties and higher diffraction limit compared with Earth-grown crystals

Role of α-Helical Structure in Organic Solvent-Activated Homodimer of Elastase Strain K

Recombinant elastase strain K overexpressed from E. coli KRX/pCon2(3) was purified to homogeneity by a combination of hydrophobic interaction chromatography and ion exchange chromatography, with a final yield of 48% and a 25-fold increase in specific activity. The purified protein had exhibited a first ever reported homodimer size of 65 kDa by SDS-PAGE and MALDI-TOF, a size which is totally distinct from that of typically reported 33 kDa monomer from P. aeruginosa. The organic solvent stability experiment had demonstrated a stability pattern which completely opposed the rules laid out in previous reports in which activity stability and enhancement were observed in hydrophilic organic solvents such as DMSO, methanol, ethanol and 1-propanol. The high stability and enhancement of the enzyme in hydrophilic solvents were explained from the view of alteration in secondary structures. Elastinolytic activation and stability were observed in 25 and 50% of methanol, respectively, despite slight reduction in α-helical structure caused upon the addition of the solvent. Further characterization experiments had postulated great stability and enhancement of elastase strain K in broad range of temperatures, pHs, metal ions, surfactants, denaturing agents and substrate specificity, indicating its potential application in detergent formulation

Geobacillus zalihae sp. nov., a thermophilic lipolytic bacterium isolated from palm oil mill effluent in Malaysia

<p>Abstract</p> <p>Background</p> <p>Thermophilic <it>Bacillus </it>strains of phylogenetic <it>Bacillus </it>rRNA group 5 were described as a new genus <it>Geobacillus</it>. Their geographical distribution included oilfields, hay compost, hydrothermal vent or soils. The members from the genus <it>Geobacillus </it>have a growth temperatures ranging from 35 to 78°C and contained iso-branched saturated fatty acids (iso-15:0, iso-16:0 and iso-17:0) as the major fatty acids. The members of <it>Geobacillus </it>have similarity in their 16S rRNA gene sequences (96.5–99.2%). Thermophiles harboring intrinsically stable enzymes are suitable for industrial applications. The quest for intrinsically thermostable lipases from thermophiles is a prominent task due to the laborious processes via genetic modification.</p> <p>Results</p> <p>Twenty-nine putative lipase producers were screened and isolated from palm oil mill effluent in Malaysia. Of these, isolate T1^Twas chosen for further study as relatively higher lipase activity was detected quantitatively. The crude T1 lipase showed high optimum temperature of 70°C and was also stable up to 60°C without significant loss of crude enzyme activity. Strain T1^Twas a Gram-positive, rod-shaped, endospore forming bacterium. On the basic of 16S rDNA analysis, strain T1^Twas shown to belong to the <it>Bacillus </it>rRNA group 5 related to <it>Geobacillus thermoleovorans </it>(DSM 5366^T) and <it>Geobacillus kaustophilus </it>(DSM 7263^T). Chemotaxonomic data of cellular fatty acids supported the affiliation of strain T1^Tto the genus <it>Geobacillus</it>. The results of physiological and biochemical tests, DNA/DNA hybridization, RiboPrint analysis, the length of lipase gene and protein pattern allowed genotypic and phenotypic differentiation of strain T1^Tfrom its validly published closest phylogenetic neighbors. Strain T1^Ttherefore represents a novel species, for which the name <it>Geobacillus zalihae </it>sp. nov. is proposed, with the type strain T1^T(=DSM 18318^T; NBRC 101842^T).</p> <p>Conclusion</p> <p>Strain T1^Twas able to secrete extracellular thermostable lipase into culture medium. The strain T1^Twas identified as <it>Geobacillus zalihae </it>T1^Tas it differs from its type strains <it>Geobacillus kaustophilus </it>(DSM 7263^T) and <it>Geobacillus thermoleovorans </it>(DSM 5366^T) on some physiological studies, cellular fatty acids composition, RiboPrint analysis, length of lipase gene and protein profile.</p

Comparison of estimation capabilities of response surface methodology (RSM) with artificial neural network (ANN) in lipase-catalyzed synthesis of palm-based wax ester

<p>Abstract</p> <p>Background</p> <p>Wax esters are important ingredients in cosmetics, pharmaceuticals, lubricants and other chemical industries due to their excellent wetting property. Since the naturally occurring wax esters are expensive and scarce, these esters can be produced by enzymatic alcoholysis of vegetable oils. In an enzymatic reaction, study on modeling and optimization of the reaction system to increase the efficiency of the process is very important. The classical method of optimization involves varying one parameter at a time that ignores the combined interactions between physicochemical parameters. RSM is one of the most popular techniques used for optimization of chemical and biochemical processes and ANNs are powerful and flexible tools that are well suited to modeling biochemical processes.</p> <p>Results</p> <p>The coefficient of determination (R²) and absolute average deviation (AAD) values between the actual and estimated responses were determined as 1 and 0.002844 for ANN training set, 0.994122 and 1.289405 for ANN test set, and 0.999619 and 0.0256 for RSM training set respectively. The predicted optimum condition was: reaction time 7.38 h, temperature 53.9°C, amount of enzyme 0.149 g, and substrate molar ratio 1:3.41. The actual experimental percentage yield was 84.6% at optimum condition, which compared well to the maximum predicted value by ANN (83.9%) and RSM (85.4%). The order of effective parameters on wax ester percentage yield were; respectively, time with 33.69%, temperature with 30.68%, amount of enzyme with 18.78% and substrate molar ratio with 16.85%, whereas R²and AAD were determined as 0.99998696 and 1.377 for ANN, and 0.99991515 and 3.131 for RSM respectively.</p> <p>Conclusion</p> <p>Though both models provided good quality predictions in this study, yet the ANN showed a clear superiority over RSM for both data fitting and estimation capabilities.</p

High yield lipase-catalyzed synthesis of Engkabang fat esters for the cosmetic industry

Engkabang fat esters were produced via alcoholysis reaction between Engkabang fat and oleyl alcohol, catalyzed by Lipozyme RM IM. The reaction was carried out in a 500 ml Stirred tank reactor using heptane and hexane as solvents. Response surface methodology (RSM) based on a four-factor-five-level Central composite design (CCD) was applied to evaluate the effects of synthesis parameters, namely temperature, substrate molar ratio (oleyl alcohol: Engkabang fat), enzyme amount and impeller speed. The optimum yields of 96.2% and 91.4% were obtained for heptane and hexane at the optimum temperature of 53.9 °C, impeller speeds of 309.5 and 309.0 rpm, enzyme amounts of 4.82 and 5.65 g and substrate molar ratios of 2.94 and 3.39:1, respectively. The actual yields obtained compared well with the predicted values of 100.0% and 91.5%, respectively. Meanwhile, the properties of the esters show that they are suitable to be used as ingredient for cosmetic applications

Crystallization of N-terminal Strep-tagged Fusion Lipase from Thermostable Bacillus sp. Strain 42.

Lipases have great potential to be used in industries due to their favourable properties such as substrate specific, enantiomerically selective, regioselective and mild reactions conditions. Lipases of microbial origin are generally more stable than lipases from animal or plant and as such they are useful source for industrial enzymes. A 1.2 kb lipase gene (AY 78735) [1], isolated from solvent stable and thermostable Bacillus sp. strain 42 was overexpressed using pET51b vector with E. coli host strain BL21(DE3) pLysS, in which the fusion lipase contains N-terminal Strep-tag II affinity tag [2]. The purified fusion lipase, at protein concentration of about 4.0 mg/mL, was induced to crystallize in 0.1 M MES buffer at pH 6.5 without the presence of salt, but in the presence of only 12% w/v PEG 20 000 as precipitant. Crystallization reactions were carried out using vapour diffusion methods at 16˚C. Crystals were formed after 12 hours incubation. The crystals with size measuring around 0.04 X 0.12 mm were shown to be heavily stained with protein dyes. Lip 42 lipase is highly homologous to three crystallized lipases from thermophilic Bacillus sp., namely T1 lipase [3], P1 lipase [4] and L1 lipase [5]. Lip 42 protein crystals, despite having almost 97% similar homology in amino acid sequence, showed a different shape and crystallization condition. The shape of Lip 42 crystal appeared to be partly attributed to the presence of N-terminal tag

Effect of nutritional factors on the growth and production of biosurfactant by Pseudomonas aeruginosa strain 181

The growth and production of biosurfactant by P. seudomonas aeruginosa (181) was dependant on nutritional factors. Among the eleven carbon sources tested, glucose supported the maximum growth (0.25 g/L) with the highest biosurfactant yield and this was followed by glycerol. Glucose reduced the surface tension to 35.3 dyne/ cm and gave an E24 reading of 62.7%. Butanol gave the lowest growth and had no biosurfactant production. For the nitrogen sources tested, casamino acid supported a growth of 0.21 g/L which reduced the surface tension to 41.1 dyne/cm and gave an E24 reading of 56%. Soytone was assimilated similarly, with good growth and high biosurfactant production. Corn steep liquor gave the lowest growth and did not show any biosurfactant activity

Construction of new genetic tools as alternatives for protein overexpression in Escherichia coli and pseudomonas aeruginosa

Background: Pseudomonas protein expression in E. coli is known to be a setback due to signifi cant genetic variation and absence of several genetic elements in E. coli for regulation and activation of Pseudomonas proteins. Modifi cations in promoter/repressor system and shuttle plasmid maintenance have made the expression of stable and active Pseudomonas protein possible in both Pseudomonas sp. and E. coli. Objectives: Construction of shuttle expression vectors for regulation and overexpression of Pseudomonas proteins in Pseudomonas sp. and E. coli. Materials and Methods: Pseudomonas-Escherichia shuttle expression vectors, pCon2(3), pCon2(3)-Kan and pCon2(3)-Zeo as well as E. coli expression vectors of pCon4 and pCon5 were constructed from pUCP19-, pSS213-, pSTBlue-1- and pPICZαAbased vectors. Protein overexpression was measured using elastase strain K as passenger enzyme in elastinolytic activity assay. Results: The integration of two series of IPTG inducible expression cassettes in pCon2(3), pCon2(3)-Kan and pCon2(3)- Zeo, each carrying an E. coli lac-operon based promoter, Plac, and a tightly regulated T7(A1/O4/O3) promoter/repressor system was performed to facilitate overexpression study of the organic solvent-tolerant elastase strain K. These constructs have demonstrated an elastinolytic fold of as high as 1464.4 % in comparison to other published constructs. pCon4 and pCon5, on the other hand, are series of pCon2(3)-derived vectors harboring expression cassettes controlled by PT7(A1/O4/O3) promoter, which conferred tight regulation and repression of basal expression due to existence of respective double operator sites, O3 and O4, and lacIq. Conclusions: The constructs off ered remarkable assistance for overexpression of heterogeneous genes in Pseudomonas sp.and E. coli for downstream applications such as in industries and structural biology study

Optimization and in silico analysis of cold-adapted lipase from an antarctic pseudomonas sp strain AMS8 reaction in Triton X-100 reverse micelles

A moderate yield of a purified enzyme can be achieved by using the simple technique of reverse micellar extraction (RME). RME is a liquid–liquid extraction method that uses a surfactant and an organic solvent to extract biomolecules. Instead of traditional chromatographic purification methods, which are tedious and expensive, RME using the nonionic surfactant Triton X-100 and toluene is used as an alternative purification technique to purify a recombinant cold-adapted lipase, AMS8. Various process parameters were optimized to maximize the activity recovery of the AMS8 lipase. The optimal conditions were found to be 50 mM sodium phosphate buffer, pH 7, 0.125 M NaCl, and 0.07 M Triton X-100 in toluene at 10 °C. Approximately 56% of the lipase activity was successfully recovered. Structural analysis of the lipase in a reverse micelle (RM) was performed using an in silico approach. The predicted model of AMS8 lipase was simulated in the Triton X-100/toluene reverse micelles from 5 to 40 °C. The lid 2 was slightly opened at 10 °C. However, the secondary structure of AMS8 was most affected in the non-catalytic domain compared to the catalytic domain, with an increased coil conformation. These results suggest that an AMS8 lipase can be extracted using Triton X-100/water/toluene micelles at low temperature. This RME approach will be an important tool for the downstream processing of recombinant cold-adapted lipase