Immobilization of mutated xylanase from Neocallimastix patriciarum in E. coli and application for kraft pulp biobleaching / Imobilização de xilanase mutada de Neocallimastix patriciarum em E. coli e aplicação para polpa kraft

Abstract As an important enzyme, xylanase is widely used in the food, pulp, and textile industry. Different applications of xylanase warrant specific conditions including temperature and pH. This study aimed to carry out sodium alginate beads as carrier to immobilize previous reported mutated xylanase from Neocallimastix patriciarum which expressed in E. coli, the activity of immobilization of mutated xylanase was elevated about 4% at pH 6 and 13% at 62 °C. Moreover, the immobilized mutated xylanase retained a greater proportion of its activity than the wide type in thermostability. These properties suggested that the immobilization of mutated xylanase has potential to apply in biobleaching industry.

Resumo Como importante enzima, a xilanase é amplamente utilizada na indústria alimentícia, de celulose e têxtil. Diferentes aplicações de xilanase garantem condições específicas, incluindo temperatura e pH. Este estudo teve como objetivo realizar grânulos de alginato de sódio como carreador para imobilizar xilanase mutada relatada anteriormente de Neocallimastix patriciarum que expressa em E. coli, a atividade de imobilização da xilanase mutada foi elevada em cerca de 4% em pH 6 e 13% a 62 °C. Além disso, a xilanase mutada imobilizada reteve uma proporção maior de sua atividade do que o tipo amplo em termoestabilidade. Essas propriedades sugerem que a imobilização da xilanase mutada tem potencial para aplicação na indústria de biobranqueamento.

Immobilization of mutated xylanase from Neocallimastix patriciarum in E coli and application for kraft pulp biobleaching / Imobilização de xilanase mutada de Neocallimastix patriciarum em E coli e aplicação para polpa kraft

As an important enzyme, xylanase is widely used in the food, pulp, and textile industry. Different applications of xylanase warrant specific conditions including temperature and pH. This study aimed to carry out sodium alginate beads as carrier to immobilize previous reported mutated xylanase from Neocallimastix patriciarum which expressed in E. coli, the activity of immobilization of mutated xylanase was elevated about 4% at pH 6 and 13% at 62 °C. Moreover, the immobilized mutated xylanase retained a greater proportion of its activity than the wide type in thermostability. These properties suggested that the immobilization of mutated xylanase has potential to apply in biobleaching industry.

Como importante enzima, a xilanase é amplamente utilizada na indústria alimentícia, de celulose e têxtil. Diferentes aplicações de xilanase garantem condições específicas, incluindo temperatura e pH. Este estudo teve como objetivo realizar grânulos de alginato de sódio como carreador para imobilizar xilanase mutada relatada anteriormente de Neocallimastix patriciarum que expressa em E. coli, a atividade de imobilização da xilanase mutada foi elevada em cerca de 4% em pH 6 e 13% a 62 °C. Além disso, a xilanase mutada imobilizada reteve uma proporção maior de sua atividade do que o tipo amplo em termoestabilidade. Essas propriedades sugerem que a imobilização da xilanase mutada tem potencial para aplicação na indústria de biobranqueamento.

Immobilization of mutated xylanase from Neocallimastix patriciarum in E. coli and application for kraft pulp biobleaching

Abstract As an important enzyme, xylanase is widely used in the food, pulp, and textile industry. Different applications of xylanase warrant specific conditions including temperature and pH. This study aimed to carry out sodium alginate beads as carrier to immobilize previous reported mutated xylanase from Neocallimastix patriciarum which expressed in E. coli, the activity of immobilization of mutated xylanase was elevated about 4% at pH 6 and 13% at 62 °C. Moreover, the immobilized mutated xylanase retained a greater proportion of its activity than the wide type in thermostability. These properties suggested that the immobilization of mutated xylanase has potential to apply in biobleaching industry.

Resumo Como importante enzima, a xilanase é amplamente utilizada na indústria alimentícia, de celulose e têxtil. Diferentes aplicações de xilanase garantem condições específicas, incluindo temperatura e pH. Este estudo teve como objetivo realizar grânulos de alginato de sódio como carreador para imobilizar xilanase mutada relatada anteriormente de Neocallimastix patriciarum que expressa em E. coli, a atividade de imobilização da xilanase mutada foi elevada em cerca de 4% em pH 6 e 13% a 62 °C. Além disso, a xilanase mutada imobilizada reteve uma proporção maior de sua atividade do que o tipo amplo em termoestabilidade. Essas propriedades sugerem que a imobilização da xilanase mutada tem potencial para aplicação na indústria de biobranqueamento.

Thermostable CRISPR/Cas9 genome editing system and its application in construction of cell factories with thermophilic bacteria: a review / 生物工程学报

The diverse thermophilic strains of Thermoanaerobacter, serving as unique platforms with a broad range of application in biofuels and chemicals, have received wide attention from scholars and practitioners. Although biochemical experiments and genome sequences have been reported for a variety of Thermoanaerobacter strains, an efficient genetic manipulation system remains to be established for revealing the biosynthetic pathways of Thermoanaerobacter. In line with this demand, the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) systems for editing, regulating and targeting genomes have been well developed in thermophiles. Here, we reviewed and discussed the current status, associated challenges, and future perspectives of the construction of thermostable CRISPR/Cas9 genome editing systems for some representative Thermoanaerobacter species. The establishment, optimization, and application of thermostable CRISPR/Cas genome editing systems would potentially provide a foundation for further genetic modification of thermophilic bacteria.

Extracellular expression in Bacillus subtilis of a thermostable Geobacillus stearothermophilus lipase

BACKGROUND The extracellular expression of enzymes in a secretion host such as Bacillus subtilis is a useful strategy in reducing the cost of downstream processing of industrial enzymes. Here, we present the first report of the successful extracellular expression in Bacillus subtilis WB800 of Geobacillus stearothermophilus lipase (T1.2RQ), a novel industriallydesirable thermostable lipolytic enzyme which has an excellent hydrolytic and transesterification activity. Signal peptides of a-amylase, extracellular protease, and lipase A, as well as two different promoters, were used in the secretion and expression of lipase T1.2RQ. RESULTS Lipase activity assay using p-nitrophenyl laurate showed that all three signal peptides directed the secretion of lipase T1.2RQ into the extracellular medium. The signal peptide of lipase A, resulted in the highest extracellular yield of 5.6 U/ml, which corresponds to a 6-fold increase over the parent Bacillus subtilis WB800 strain. SDS-PAGE and zymogram analysis confirmed that lipase T1.2RQ was correctly processed and secreted in its original size of 44 kDa. A comparison of the expression levels of lipase T1.2RQ in rich medium and minimal media showed that the enzyme was better expressed in rich media, with up to an 8-fold higher yield over minimal media. An attempt to further increase the lipase expression level by promoter optimization showed that, contrary to expectation, the optimized promoter exhibited similar expression levels as the original one, suggesting the need for the optimization of downstream factors. CONCLUSIONS The successful extracellular secretion of lipase T1.2RQ in Bacillus subtilis represents a remarkable feat in the industrial-scale production of this enzyme

Structural-functional analyses of textile dye degrading azoreductase, laccase and peroxidase: a comparative in silico study

Background: Textile industry not only plays a vital role in our daily life but also a prominent factor in improving global economy. One of the environmental concern is it releases huge quantities of toxic dyes in the water leading to severe environmental pollution. Bacterial laccase and azoreductase successfully oxidize complex chemical structure of nitrogen group-containing azo dyes. Additionally, the presence of textile dye infuriates bacterial peroxidase to act as a dye degrading enzyme. Our present study deals with three textile dye degrading enzymes laccase, azoreductase, and peroxidase through analyzing their structural and functional properties using standard computational tools. Result: According to the comparative analysis of physicochemical characteristics, it was clear that laccase was mostly made up of basic amino acids whereas azoreductase and peroxidase both comprised of acidic amino acids. Higher aliphatic index ascertained the thermostability of all these three enzymes. Negative GRAVY value of the enzymes confirmed better water interaction of the enzymes. Instability index depicted that compared to laccase and preoxidase, azoreductase was more stable in nature. It was also observed that the three model proteins had more than 90% of total amino acids in the favored region of Ramachandran plot. Functional analysis revealed laccase as multicopper oxidase type enzyme and azoreductase as FMN dependent enzyme, while peroxidase consisted of α-ß barrel with additional haem group. Conclusion: Present study aims to provide knowledge on industrial dye degrading enzymes, choosing the suitable enzyme for industrial set up and to help in understanding the experimental laboratory requirements as well.

Paenibacillus dendritiformis DDS2: A new member to alkaline-thermophilic protease producers

@#Aim: Most of the industrial processes require thermostable alkaline proteases. Thus, a search was initiated to isolate and characterize a bacterium which can produce thermostable alkaline protease. Methodology and results: Best higher titer thermostable alkaline protease producing wild type organism was screened from beef, dog and fish decaying soil samples. Among the 92 bacterial strains, three strains which produced alkaline proteases having activities at pH 10.5 and above 70 °C were selected. Among the three strains, the one from the dog decaying soil (Strain DDS2 ; DDS-dog decaying soil) which produced the protease showing highest activity at pH 10.5 and 73 °C and stability (half-life: 10.5 h) without additives was selected and identified. Based on the biochemical and morphological studies, strain DDS2 could be either Paenibacillus dendritiformis or P. thiaminolyticus. From 16S rDNA sequencing, the strain DDS2 was confirmed as P. dendritiformis. Conclusion, significance and impact: This is the first report published to show that P. dendritiformis is a protease producer and the organism was named as P. dendritiformis DDS2.

Screening, Production and Partial Characterization of a Thermostable Laccase from Trametes sp. Isolate B7 with Biotechnological Potentials

The search for efficient and green oxidation technologies has increased interest in utilization of laccases in non conventional methods. Laccases catalyze a wide range of substrates due to low substrate specificity and strong oxidative potentials. Challenges to large-scale enzyme utilization include, low enzyme activity and instability which restrict use in many areas of biotechnology. In the study, 59 fungi comprising Aspergillus niger (40%), Trichoderma harzianum (31%), Aspergillus flavus (9.0%), Trichoderma viride (5.0%), Fusarium oxysporum (5.0%), Rhizopus stolonifer (5.0%), Trametes sp. (3.0%) and Aspergillus nidulans (2.0%) were isolated and screened for laccase production. Plate screening test showed 57.5%, 34.0% and 8.5% of fungi were laccase-positive on ABTS, Guaiacol, and α-naphthol agar respectively. Isolates were further screened in liquid cultures, and the highest laccase producer identified molecularly. Trametes sp isolate B7 was selected for solid state fermentation (SSF). Laccase production in SSF was highest at pH 5.0 (2356 U/mL). The purified laccase showed high activity (pH 3.0 - 6.0) and stability (pH 3.0 - 8.5) using ABTS. It was active (20 - 80°C) and thermostable (30 - 80°C) with optimum stability at 70°C (100% for 1 hour). The percentage decolourization of Phenol red were 28% and 36% using 1000 U/mL and 2000 U/mL crude laccases respectively. Similarly, RBBR (100%), Congo red (75%) and Malachite green (62%), and 77.4%, 64% and 28% were decolourized using 1000 U/mL and 2000 U/mL crude laccases respectively. ABTS agar was very reliable in large-scale screening for laccase which possessed thermostable property and degraded synthetic dyes without use of enzyme mediators. These attribute made the enzyme suitable for application in industry and biotechnology.

Screening of bacterial extracellular xylanase producers with potential for cellulose pulp biobleaching

In this study, two hundred fifty-seven bacterial isolates from a suppressive soil library were screened to study their secretion of alkali-thermostable xylanases for potential use in cellulose pulp biobleaching. Xylanase activity was evaluated in solid and liquid media using xylan as the carbon source. Isolates were initially evaluated for the degradation of xylan in solid media by the congo red test. Selected strains were evaluated in liquid media for enzymatic activity and determination of total protein concentration using a crude protein extract (CPE). An isolate identified as Bacillus species TC-DT13 produced the highest amount of xylanase (1808 U mL-1). The isolate was active and stable at 70°C and pH 9.0, conditions which are necessary for the paper industry. This isolate can grow and produce xylanase in medium containing wheat fiber as a substrate. The CPE of this isolate was used in preliminary testing on cellulose pulp bleaching; enzyme treatment of the pulp resulted in a 5% increase of whiteness.

Characterization and structure of a novel thermostable glucoamylase from Talaromyces leycettanus JCM12802 / 生物工程学报

Glucoamylase is a critical ingredient for saccharification in the starch decomposition, and widely used in food, pharmaceutical and fermentation industries. Glucoamylases are usually thermostable and have peak activities at high temperature, as required for the industrial process of glucose production. In this study, a glucoamylase gene belonging to the glycoside hydrolase (GH) family 15, Tlga15A, was cloned from Talaromyces leycettanus JCM12802, and successfully expressed in Pichia pastoris GS115. Recombinant glucoamylase TlGA showed optimal activities at pH 4.5 and 75 °C. The result of thermostability analysis showed that TlGA retained above 70% activity after incubating for 1 h at 65 °C, and 43% residual activity after 30 min at 70 °C. Moreover, TlGA had high resistance to most metal ions and chemical reagents tested. Various starch substrates could be hydrolyzed by TlGA, including soluble starch (255.6±15.3) U/mg, amylopectin (342.3±24.7) U/mg, glycogen (185.4±12.5) U/mg, dextrin (423.3±29.3) U/mg and pullulan (65.7±8.1) U/mg. The primary, secondary and tertiary structures of glucoamylase were further analyzed. The low ratio of Gly in the primary structure and low exposed nonpolarity solvent accessible surface in the tertiary structure may be the main reasons for TlGA's thermostability. These results show that TlGA is great promising for potential use in the commercial production of glucose syrups. Moreover, this research will provide knowledge and innovating ideas for the improvement of glucoamylase thermostability.

Recombinant production and biochemical and in silico characterization of lactate dehydrogenase from Geobacillus thermodenitrificans DSM-465

Background: Lactate dehydrogenase (LDH) is an enzyme of glycolytic pathway, ubiquitously found in living organisms. Increased glycolysis and LDH activity are associated with many pathologic conditions including inflammation and cancer, thereby making the enzyme a suitable drug target. Studies on conserved structural and functional domains of LDH from various species reveal novel inhibitory molecules. Our study describes Escherichia coli production and characterization of a moderately thermostable LDH (LDH-GT) from Geobacillus thermodenitrificans DSM-465. An in silico 3D model of recombinant enzyme and molecular docking with a set of potential inhibitors are also described. Results: The recombinant enzyme was overexpressed in E. coli and purified to electrophoretic homogeneity. The molecular weight of the enzyme determined by MALDI-TOF was 34,798.96 Da. It exhibited maximum activity at 65°C and pH 7.5 with a KM value for pyruvate as 45 µM. LDH-GT and human LDH-A have only 35.6% identity in the amino acid sequence. On the contrary, comparison by in silico structural alignment reveals that LDH-GT monomer has approximately 80% identity to that of truncated LDH-A. The amino acids "GEHGD" as well as His179 and His193 in the active site are conserved. Docking studies have shown the binding free energy changes of potential inhibitors with LDH-A and LDH-GT ranging from −407.11 to −127.31 kJ mol−1 . Conclusions: By highlighting the conserved structural and functional domains of LDH from two entirely different species, this study has graded potential inhibitory molecules on the basis of their binding affinities so that they can be applied for in vivo anticancer studies

Effect of ultraviolet radiation on physiological and biochemical properties of yeast Saccharomyces cerevisiae during fermentation of ultradispersed starch raw material

Background: Study of correlation between pretreatment of yeast with ultraviolet radiation and efficiency of further fermentation of wort made of ultrafine grain particles to ethanol. Results: We investigated three races of industrial yeast Saccharomyces cerevisiae (native and irradiated by ultraviolet). Physiological properties during fermentation of starchy wort were tested in all variants. It was shown that activation of the yeast by ultraviolet radiation allows to further increase the ethanol yield by 25% on average compared with the native yeast races when using thin (up to micro- and nano-sized particles) or standard grain grinding. Conclusions: Using mechanical two-stage grinding of starchy raw materials and ultraviolet pretreatment of yeast, the efficiency of saccharification of starch and fermentation of wort to ethanol was increased.

A new isolate of thermophilic and organic solvent tolerant bacteria for lipase production using basal medium of palm kernel cake 80-87

@#Aims: This research focused on the selection of potential strains especially bacteria that can grow effectively in palm kernel cake (PKC) and produce high amount of thermostable and solvent tolerant (TS-OST) lipase. The work involved the exploration of renewable PKC as potential fermentation medium for discovery to novel TS-OST lipase that would have excellent tolerance and activity in presence of organic solvents with high temperatures for industrial applications.Methodology and results: Using palm kernel cake (PKC) as source of thermophilic bacteria, 53 bacterial strains were found survived at temperature 65 °C. However, after subcultured several times, only 17 strains were found as pure thermophilic strains. Preliminary screening both qualitative and quantitative was performed to all 17 potential thermophilic bacterial strains and showed that only 11 purified thermophilic strains are lipase producer. Strain PKC-P1 produced highest enzyme activity (11.13 U/g), followed by PKC-P13 and PKC-C9. The lowest enzymeactivity was lipase produced byPKC-C10 (0.76U/g). Strain PKC-P1 has been classified as Gram negative bacteria and identified as Bacillus smithiistrain PKC_P1.Conclusion, significance and impact of study: PKC as a by-product of oil palm industry consistsof many nutrients that can give benefits towards industry and can be utilized in order to produce enzymes like lipases. From these results, it could be concluded that this lipase stable at temperature 65 °C and pH 7 and may be a potential candidate to be used in a variety of biotechnological applications. This finding revealed that a bacterial strain obtained from oil-rich environment which is PKC through isolation process has potential as a source of more economical enzyme to be applied in biotechnology industr

Thermostable chitinase from Cohnella sp. A01: isolation and product optimization

Abstract Twelve bacterial strains isolated from shrimp farming ponds were screened for their growth activity on chitin as the sole carbon source. The highly chitinolytic bacterial strain was detected by qualitative cup plate assay and tentatively identified to be Cohnella sp. A01 based on 16S rDNA sequencing and by matching the key morphological, physiological, and biochemical characteristics. The cultivation of Cohnella sp. A01 in the suitable liquid medium resulted in the production of high levels of enzyme. The colloidal chitin, peptone, and K2HPO4 represented the best carbon, nitrogen, and phosphorus sources, respectively. Enzyme production by Cohnella sp. A01 was optimized by the Taguchi method. Our results demonstrated that inoculation amount and temperature of incubation were the most significant factors influencing chitinase production. From the tested values, the best pH/temperature was obtained at pH 5 and 70 °C, with Km and V max values of chitinase to be 5.6 mg/mL and 0.87 µmol/min, respectively. Ag+, Co2+, iodoacetamide, and iodoacetic acid inhibited the enzyme activity, whereas Mn2+, Cu2+, Tweens (20 and 80), Triton X-100, and EDTA increased the same. In addition, the study of the morphological alteration of chitin treated by enzyme by SEM revealed cracks and pores on the chitin surface, indicating a potential application of this enzyme in several industries.

Process optimization for production and purification of a thermostable, organic solvent tolerant lipase from Acinetobacter sp. AU07

ABSTRACT The purpose of this study was to isolate, purify and optimize the production conditions of an organic solvent tolerant and thermostable lipase from Acinetobacter sp. AU07 isolated from distillery waste. The lipase production was optimized by response surface methodology, and a maximum production of 14.5 U/mL was observed at 30 ºC and pH 7, using a 0.5% (v/v) inoculum, 2% (v/v) castor oil (inducer), and agitation 150 rpm. The optimized conditions from the shake flask experiments were validated in a 3 L lab scale bioreactor, and the lipase production increased to 48 U/mL. The enzyme was purified by ammonium sulfate precipitation and ion exchange chromatography and the overall yield was 36%. SDS-PAGE indicated a molecular weight of 45 kDa for the purified protein, and Matrix assisted laser desorption/ionization time of flight analysis of the purified lipase showed sequence similarity with GDSL family of lipases. The optimum temperature and pH for activity of the enzyme was found to be 50 ºC and 8.0, respectively. The lipase was completely inhibited by phenylmethylsulfonyl fluoride but minimal inhibition was observed when incubated with ethylenediaminetetraacetic acid and dithiothreitol. The enzyme was stable in the presence of non-polar hydrophobic solvents. Detergents like SDS inhibited enzyme activity; however, there was minimal loss of enzyme activity when incubated with hydrogen peroxide, Tween 80 and Triton X-100. The kinetic constants (Km and Vmax) revealed that the hydrolytic activity of the lipase was specific to moderate chain fatty acid esters. The Vmax, Km and Vmax/Km ratio of the enzyme were 16.98 U/mg, 0.51 mM, and 33.29, respectively when 4-nitrophenyl palmitate was used as a substrate.

Purification and characterization of extracellular α-amylase from a thermophilic Anoxybacillus thermarum A4 strain

ABSTRACT α-Amylase from Anoxybacillus thermarum A4 was purified using ammonium sulphate precipitation and Sephadex G-100 gel filtration chromatography, with 29.8-fold purification and 74.6% yield. A4 amylase showed best performance for soluble potato starch hydrolysis at 70 °C and pH 5.5-10.5. A4 amylase was extremely stable at +4 °C, and the enzyme retained over 65% of its original α-amylase activity at 70 °C and 43% at 90 °C. The enzyme's Km values for soluble starch, amylopectin and amylose substrates were obtained as 0.9, 1.3 and 0.5 mg/mL, respectively. EDTA, Hg2+, B4O7 2-, OH-, CN- , and urea exhibited different inhibition effects; their IC50 values were identified as 8.0, 5.75, 16.5, 15.2, 8.2 and 10.9 mM, respectively. A4 amylase exhibited extreme stability toward some surfactants and perfect match for a wide variety of commercial solid and liquid detergents at 55 °C. So, it may be considered to be potential applications for detergent and other industrial uses.

The genotypic diversity and lipase production of some thermophilic bacilli from different genera

Abstract Thermophilic 32 isolates and 20 reference bacilli were subjected to Rep-PCR and ITS-PCR fingerprinting for determination of their genotypic diversity, before screening lipase activities. By these methods, all the isolates and references could easily be differentiated up to subspecies level from each other. In screening assay, 11 isolates and 7 references were found to be lipase producing. Their extracellular lipase activities were measured quantitatively by incubating in both tributyrin and olive oil broths at 60 °C and pH 7.0. During the 24, 48 and 72-h period of incubation, the changes in the lipase activities, culture absorbance, wet weight of biomass and pH were all measured. The activity was determined by using pNPB in 50 mM phosphate buffer at pH 7.0 at 60 °C. The lipase production of the isolates in olive oil broths varied between 0.008 and 0.052, whereas these values were found to be 0.002-0.019 (U/mL) in the case of tyributyrin. For comparison, an index was established by dividing the lipase activities to cell biomass (U/mg). The maximum thermostable lipase production was achieved by the isolates F84a, F84b, and G. thermodenitrificans DSM 465T (0.009, 0.008 and 0.008 U/mg) within olive oil broth, whereas G. stearothermophilus A113 displayed the highest lipase activity than its type strain in tyributyrin. Therefore, as some of these isolates displayed higher activities in comparison to references, new lipase producing bacilli were determined by presenting their genotypic diversity with DNA fingerprinting techniques.

Molecular characterization of a novel thermostable laccase PPLCC2 from the brown rot fungus Postia placenta MAD-698-R

Background Laccase has been considered important for the degradation of lignocellulose by wood rot fungi. The properties and functions of laccase in white rot fungi have been investigated extensively, but those from brown rot fungi remain largely unknown. In this paper, a laccase isoform Pplcc2 from the brown rot fungus Postia placenta MAD-698-R was expressed heterologously in Pichia pastoris GS115, purified and the properties of the enzyme were determined. Results The molecular weight of the protein was determined to be 67 kDa using SDS-PAGE. It cannot oxidize syringaldazine (SGZ), but it can oxidize 2,2'-azino-di-(3-ethylbenzothialozin-6-Sulfonic acid) (ABTS) and 2,6-dimethoxyphenol (DMP). Specific activity for ABTS was 1960 ± 19 Unit/mg. The catalytic constant (k cat) was 1213 ± 18.3 s-1 for ABTS and 293.2 ± 21.9 s-1 for DMP. Km was 22.08 µM for ABTS and 11.62 µM for DMP. The optimal pH for the oxidation of ABTS and DMP was 3.5 and 5.0 respectively. The optimal temperature for the oxidation of ABTS and DMP was 60°C. Conclusions This is the first identified thermo activated and thermostable laccase in brown rot fungi. This investigation will contribute to understanding the roles played by laccases in brown rot fungi.

Enhanced production of thermostable lipase from Bacillus cereus ASSCRC-P1 in waste frying oil based medium using statistical experimental design.

This study aim to isolate, identify a bacterial isolate and optimize production medium using frying oil waste for lipase production. Nine strains were isolated from an Egyptian soil samples. Among the isolates, a potent bacterial candidate ASSCRC-P1 was found to be the most potent lipase producer strain at 60 °C. Genotypic identification of ASSCRC-P1 showed 94% similarity with Bacillus sp. strains. Phylogenetic tree confirmed that ASSCRC-P1 was nearly similar to Bacillus cereus. Therefore, it was given the name Bacillus cereus ASSCRC-P1 and its 16S rRNA nucleotide has been deposited in the GenBank Data Library under the accession number: KJ531440. A sequential optimization strategy, based on statistical experimental designs, was employed to enhance the lipase production by this strain. A 2-level Plackett–Burman design was applied to differentiate between the bioprocess parameters that significantly influence lipase production followed by Box-Behnken design to optimize the amounts of variables which have the highest positive significant effect on lipase production. Overall more than 2.15-fold improvement in lipase production was achieved due to optimization compared to that obtained using the basal medium.

Cloning and characterization of newly isolated lipase from Enterobacter sp. Bn12

A mesophilic Enterobacter sp. Bn12 producing an alkaline thermostable lipase was isolated from soil in Tehran, Iran. The lipase gene (ELBn12) was identified from a genomic library. Sequence analysis of the DNA fragment revealed an open reading frame of 879 bp encoding a lipase with a molecular mass of 31.3 kDa. The deduced amino acid sequence showed 96% identity with a lipase of Enterobacter sp. Ag1 and the identity of their DNA sequences was 88.9%. ELBn12 belongs to the lipase subfamily I.1 and its catalytic triad consists of Ser82, Asp237 and His259. The lipase was expressed in Escherichia coli (BL21) pLysS and partially purified by anion exchange chromatography. The maximum activity of ELBn12 was obtained at temperature of 60 °C and pH 8.0 towards tricaprylin (C8) and its specific activity was around 2900 U/mg. ELBn12 was stable within a broad pH range from 6.0 to 11.0. The enzyme showed high stability in both polar and nonpolar organic solvents at 50% (v/v). The lipase activity was enhanced in the presence of 10 mM of Ca2+, Mg2+ and K+, while heavy metals (Fe3+ and Zn2+) had strong inhibitory effect. ELBn12 showed high activity in the presence of 1% (w/v) nonionic surfactants, however ionic surfactants inhibited the lipolytic activity. ELBn12 characteristics show that it has a potential to be used in various industrial processes.