Purification and identification of a surfactin biosurfactant and engine oil degradation by Bacillus velezensis KLP2016.

Engine oil used in automobiles is a threat to soil and water due to the recalcitrant properties of its hydrocarbons. It pollutes surrounding environment which affects both flora and fauna. Microbes can degrade hydrocarbons containing engine oil and utilize it as a substrate for their growth. Our results demonstrated that cell-free broth of Bacillus velezensis KLP2016 (Gram + ve, endospore forming; Accession number KY214239) recorded an emulsification index (E24%) from 52.3% to 65.7% against different organic solvents, such as benzene, pentane, cyclohexane, xylene, n-hexane, toluene and engine oil. The surface tension of the cell-free broth of B. velezensis grown in Luria-Bertani broth at 35 °C decreased from 55 to 40 mN m-1at critical micelle concentration 17.2 µg/mL. The active biosurfactant molecule of cell-free broth of Bacillus velezensis KLP2016 was purified by Dietheylaminoethyl-cellulose and size exclusion chromatography, followed by HPLC (RT = 1.130), UV-vis spectrophotometry (210 nm) and thin layer chromatography (Rf = 0.90). The molecular weight of purified biosurfactant was found to be ~ 1.0 kDa, based on Electron Spray Ionization-MS. A concentration of 1980 × 10-2 parts per million of CO2 was trapped in a KOH solution after 15 days of incubation in Luria-Bertani broth containing 1% engine oil. Our results suggest that bacterium Bacillus velezensis KLP2016 may promise a new dimension to solving the engine oil pollution problem in near future.

Lipopeptide(s) associated with human microbiome as potent cancer drug.

Human microbiota comprises of trillions of microbes which have evolved with and continued to live on/ within their human hosts. Different environmental factors and diet have a large impact upon human microbiota population. These microorganisms live in synergy with their hosts and are beneficial to the host in many different ways. Many microorganisms help to fight against human diseases. Cancer is one such diseases which effects a large human population often leading to death. Cancer is also one of the most fatal human diseases killing millions of people world-wide every year. Though many treatment procedures are available but none is 100 % effective in curing cancer. In this review, we seek to understand the role of human microbiota in cancer treatment. Lipopeptide(s) (LPs) produced by different microorganisms can act as efficient drug(s) against cancer. LPs are low molecular weight lipo-proteins that are also known for their anti-cancer activities. As human microbiota belongs to an environment within the host body, a drug prepared using these microorganisms will be easily accepted by the body. This novel approach of using LPs produced by human microbiota can be considered for the much needed change in cancer treatment. Therefore, it is proposed that research should focus on the host-microbe interaction which could pave the way in understanding role played by these microorganisms in cancer treatment.

Phosphatidylserine: A cancer cell targeting biomarker.

Cancer is a leading cause of mortality and morbidity globally. Many prominent cancer-associated molecules have been identified over the recent years which include EGFR, CD44, TGFbRII, HER2, miR-497, NMP22, BTA, Fibrin/FDP etc. These biomarkers are often used for screening, detection, diagnosis, prognosis, prediction and monitoring of cancer development. Phosphatidylserine (PS) is an essential component in all human cells which is present on the inner leaflet of the cell membrane. The oxidative stress causes exposure of PS on the surface of the vascular endothelium in the cancer cells (lung, breast, pancreatic, bladder, skin, brain metastasis, rectal adenocarcinoma etc.) but not on the normal cells. The external PS is regulated by calcium-dependent flippase activity. Cancer cell lines with high surface PS have low flippase activity and high intracellular calcium content. Human Annexin-V, PS targeting antibodies (PGN635 and bavituximab and mch1N11), lysosomal protein, phospholipid Saposin C dioleoylphosphatidylserine (SapC-DOPS), peptide-peptoid hybrid PPS1, PS-binding 14-mer peptide (PSBP-6) and hexapeptide (E3) have been reported to target PS present on cancer cell surface. High expression of CD47 inhibits tumor cell phagocytosis by macrophages. The PS cancer biomarker has also been used to target the drugs to cancer cells specifically without affecting other healthy cells. Currently, the fusion protein (FP) consisting of L-methionase linked to human Annexin-V has been reported to target the cancer cells. The FP catalyzes the conversion of non-toxic prodrug selenomethionine into toxic methyl selenol which thus also prevents the methionine (essential amino acid) supplementation to the cancer cells.

Molecular characterization and bioinformatics studies of a lipase from Bacillus thermoamylovorans BHK67.

A bacterium isolated from a hot-water spring identified as Bacillus thermoamylovorans BHK67 successfully produced a thermotolerant extracellular alkaliphilic lipase. The lipase was purified to homogeneity by anion exchange chromatography with 15-fold purification and 12.1% yield. The lipase appeared to be a hexameric protein as it possessed a single band of Mr 25kDa in SDS PAGE and 150kDa in Native PAGE. DLS analysis of purified Bacillus thermoamylovorans BHK67 lipase (BTL) also showed the molecular integrity, homogeneity and stability of the enzyme. The purified lipase showed maximum activity at pH 7.5 with a half-life of 10.5h at 55°C. Kinetic study of purified lipase by Lineweaver-Burk plot provided Km (7.7mM),Vmax (90.9U/mL/min),Kcat (227.3s-1) and Kspec (29.4mMs-1) for substrate p-nitrophenylpalmitate.The purified lipase also showed astonishing stability following exposure to ethanol, n-propanol, iso-propanol, n-butanol and DMSO. Amino acid characterization of BTL by MALDI-TOF-MS showed considerable resemblance with lysophospholipase L1 related esterase of Lactobacillus ozensis DSM 23829. Experimental coupled molecular modeling postulated a structure-activity correlation of BTL as a probable contender in degradation of xenobiotic compounds, biocatalysis, biotransformation of compounds, synthesis of optically active compounds, foodstuff industry, anticancer therapeutics etc.

Lead Phytochemicals for Anticancer Drug Development.

Cancer is a serious concern at present. A large number of patients die each year due to cancer illnesses in spite of several interventions available. Development of an effective and side effects lacking anticancer therapy is the trending research direction in healthcare pharmacy. Chemical entities present in plants proved to be very potential in this regard. Bioactive phytochemicals are preferential as they pretend differentially on cancer cells only, without altering normal cells. Carcinogenesis is a complex process and includes multiple signaling events. Phytochemicals are pleiotropic in their function and target these events in multiple manners; hence they are most suitable candidate for anticancer drug development. Efforts are in progress to develop lead candidates from phytochemicals those can block or retard the growth of cancer without any side effect. Several phytochemicals manifest anticancer function in vitro and in vivo. This article deals with these lead phytomolecules with their action mechanisms on nuclear and cellular factors involved in carcinogenesis. Additionally, druggability parameters and clinical development of anticancer phytomolecules have also been discussed.

Ascorbyl palmitate synthesis in an organic solvent system using a Celite-immobilized commercial lipase (Lipolase 100L).

Ascorbyl palmitate was synthesized using a Celite-immobilized commercial lipase (Lipolase 100L) in dimethylsulfoxide (DMSO) as an organic solvent system. Lipase immobilized by surface adsorption onto Celite 545 matrix and subsequently exposed to 1 % glutaraldehyde showed 75 % binding of protein. The Celite-bound lipase was optimally active at 75 °C and pH 8.5 under shaking and showed maximum hydrolytic activity toward p-NPP as a substrate. The bound lipase was found to be stimulated only in the presence of Al3+ and EDTA. All surfactants (Tween-20, Tween-80 and Triton X-100) had an inhibitory effect on lipase activity. The optimization of various reaction conditions of ascorbyl palmitate was achieved considering one factor at a time. The esterification of ascorbic acid and palmitic acid was carried out with 1 M ascorbic acid and 2.5 M palmitic acid in DMSO at 75 °C for 18 h under shaking (120 rpm). Molecular sieves had an important effect on the ester synthesis resulting in an enhanced yield. The by-product (H2O) produced in the reaction was scavenged by the molecular sieves (20 mg/ml) added in the reaction mixture which enhanced the ester yield to 80 %. The characterization of synthesized ester was done through FTIR spectroscopy.

Purification and Characterization of an Extracellular Cholesterol Oxidase of Bacillus subtilis Isolated from Tiger Excreta.

A mesophilic Bacillus sp. initially isolated from tiger excreta and later identified as a Bacillus subtilis strain was used to produce an extracellular cholesterol oxidase (COX) in cholesterol-enriched broth. This bacterial isolate was studied for the production of COX by manipulation of various physicochemical parameters. The extracellular COX was successfully purified from the cell-free culture broth of B. subtilis by successive salting out with ammonium sulfate, dialysis, and riboflavin-affinity chromatography. The purified COX was characterized for its molecular mass/structure and stability. The enzyme possessed some interesting properties such as high native Mr (105 kDa), multimeric (pentamer of ∼21 kDa protein) nature, organic solvent compatibility, and a half-life of ∼2 h at 37 °C. The bacterial COX exhibited ∼22 % higher activity in potassium phosphate buffer (pH 7.5) in the presence of a nonionic detergent Triton X-100 at 0.05 % (v/v). The K m and V max value of COX of B. subtilis COX were found to be 3.25 mM and 2.17 µmol min ml(-1), respectively. The purified COX showed very little cytotoxicity associated with it.

Gallic acid-based alkyl esters synthesis in a water-free system by celite-bound lipase of Bacillus licheniformis SCD11501.

Gallic acid (3, 4, 5- trihydroxybenzoic acid) is an important antioxidant, anti-inflammatory, and radical scavenging agent. In the present study, a purified thermo-tolerant extra-cellular lipase of Bacillus licheniformis SCD11501 was successfully immobilized by adsorption on Celite 545 gel matrix followed by treatment with a cross-linking agent, glutaraldehyde. The celite-bound lipase treated with glutaraldehyde showed 94.8% binding/retention of enzyme activity (36 U/g; specific activity 16.8 U/g matrix; relative increase in enzyme activity 64.7%) while untreated matrix resulted in 88.1% binding/retention (28.0 U/g matrix; specific activity 8.5 U/g matrix) of lipase. The celite-bound lipase was successfully used to synthesis methyl gallate (58.2%), ethyl gallate (66.9%), n-propyl gallate (72.1%), and n-butyl gallate (63.8%) at 55(o) C in 10 h under shaking (150 g) in a water-free system by sequentially optimizing various reaction parameters. The low conversion of more polar alcohols such as methanol and ethanol into their respective gallate esters might be due to the ability of these alcohols to severely remove water from the protein hydration shell, leading to enzyme inactivation. Molecular sieves added to the reaction mixture resulted in enhanced yield of the alkyl ester(s). The characterization of synthesised esters was done through fourier transform infrared (FTIR) spectroscopy and (1) H NMR spectrum analysis.

Lipopeptides as the antifungal and antibacterial agents: applications in food safety and therapeutics.

A lot of crops are destroyed by the phytopathogens such as fungi, bacteria, and yeast leading to economic losses to the farmers. Members of the Bacillus genus are considered as the factories for the production of biologically active molecules that are potential inhibitors of growth of phytopathogens. Plant diseases constitute an emerging threat to global food security. Many of the currently available antimicrobial agents for agriculture are highly toxic and nonbiodegradable and thus cause extended environmental pollution. Moreover, an increasing number of phytopathogens have developed resistance to antimicrobial agents. The lipopeptides have been tried as potent versatile weapons to deal with a variety of phytopathogens. All the three families of Bacillus lipopeptides, namely, Surfactins, Iturins and Fengycins, have been explored for their antagonistic activities towards a wide range of phytopathogens including bacteria, fungi, and oomycetes. Iturin and Fengycin have antifungal activities, while Surfactin has broad range of potent antibacterial activities and this has also been used as larvicidal agent. Interestingly, lipopeptides being the molecules of biological origin are environmentally acceptable.

L-methionase: a therapeutic enzyme to treat malignancies.

Cancer is an increasing cause of mortality and morbidity throughout the world. L-methionase has potential application against many types of cancers. L-Methionase is an intracellular enzyme in bacterial species, an extracellular enzyme in fungi, and absent in mammals. L-Methionase producing bacterial strain(s) can be isolated by 5,5'-dithio-bis-(2-nitrobenzoic acid) as a screening dye. L-Methionine plays an important role in tumour cells. These cells become methionine dependent and eventually follow apoptosis due to methionine limitation in cancer cells. L-Methionine also plays an indispensable role in gene activation and inactivation due to hypermethylation and/or hypomethylation. Membrane transporters such as GLUT1 and ion channels like Na(2+), Ca(2+), K(+), and Cl(-) become overexpressed. Further, the α-subunit of ATP synthase plays a role in cancer cells growth and development by providing them enhanced nutritional requirements. Currently, selenomethionine is also used as a prodrug in cancer therapy along with enzyme methionase that converts prodrug into active toxic chemical(s) that causes death of cancerous cells/tissue. More recently, fusion protein (FP) consisting of L-methionase linked to annexin-V has been used in cancer therapy. The fusion proteins have advantage that they have specificity only for cancer cells and do not harm the normal cells.

Organic solvent tolerant lipases and applications.

Lipases are a group of enzymes naturally endowed with the property of performing reactions in aqueous as well as organic solvents. The esterification reactions using lipase(s) could be performed in water-restricted organic media as organic solvent(s) not only improve(s) the solubility of substrate and reactant in reaction mixture but also permit(s) the reaction in the reverse direction, and often it is easy to recover the product in organic phase in two-phase equilibrium systems. The use of organic solvent tolerant lipase in organic media has exhibited many advantages: increased activity and stability, regiospecificity and stereoselectivity, higher solubility of substrate, ease of products recovery, and ability to shift the reaction equilibrium toward synthetic direction. Therefore the search for organic solvent tolerant enzymes has been an extensive area of research. A variety of fatty acid esters are now being produced commercially using immobilized lipase in nonaqueous solvents. This review describes the organic tolerance and industrial application of lipases. The main emphasis is to study the nature of organic solvent tolerant lipases. Also, the potential industrial applications that make lipases the biocatalysts of choice for the present and future have been presented.

Peroxidase(s) in environment protection.

Industrial discharges of untreated effluents into water bodies and emissions into air have deteriorated the quality of water and air, respectively. The huge amount of pollutants derived from industrial activities represents a threat for the environment and ecologic equilibrium. Phenols and halogenated phenols, polycyclic aromatic hydrocarbons (PAH), endocrine disruptive chemicals (EDC), pesticides, dioxins, polychlorinated biphenyls (PCB), industrial dyes, and other xenobiotics are among the most important pollutants. Peroxidases are enzymes that are able to transform a variety of compounds following a free radical mechanism, thereby yielding oxidized or polymerized products. The peroxidase transformation of these pollutants is accompanied by a reduction in their toxicity, due to loss of biological activity, reduction in the bioavailability, or the removal from aqueous phase, especially when the pollutant is found in water. The review describes the sources of peroxidases, the reactions catalyzed by them, and their applications in the management of pollutants in the environment.

Improved production of L-asparaginase by Bacillus brevis cultivated in the presence of oxygen-vectors.

The efficiency of three oxygen-vectors liquid paraffin, silicone oil and n-dodecane in the production of L-asparaginase by Bacillus brevis was evaluated at 4% (v/v) concentration. All of the three oxygen-vectors were found to exhibit a stimulatory effect on L-asparaginase activity. Liquid paraffin at 6% (v/v) resulted in 34% increase in the L-asparaginase activity accompanied by a 48% increase in the production of cell mass at a 10 L scale. This improvement in L-asparaginase activity and cell mass production in the presence of liquid paraffin can be related to the fact that liquid paraffin was capable of maintaining dissolved O2 concentration above 28% through out the course of the fermentation. Maintenance of the dissolved O2 concentration above 28% could be viewed in terms of an adequate oxygen supply to the rapidly dividing cells of the bacterium, which in turn resulted in enhancement in cell mass production and l-asparaginase activity.

Synthesis of ethyl ferulate in organic medium using celite-immobilized lipase.

In the present work we have evaluated synthesis of ethyl ferulate by the esterification reaction of ferulic acid and ethanol catalyzed by a commercial lipase (Steapsin) immobilized onto celite-545 in a short period of 6h in DMSO. The immobilized lipase was treated with cross-linking agent glutaraldehyde (1%; v/v). The optimum synthesis of ethyl ferulate was recorded at 45°C, pH 8.5 and 1:1 ratio of ethanol and ferulic acid. Co(2+), Ba(2+)and Pb(2+) ions enhanced the synthesis of ethyl ferulate Hg(2+), Cd(3+)and NH(4+) ions had mild inhibitory effect. The celite-bound lipase produced 68 mM of ethyl ferulate under optimized reaction conditions.

Glutaraldehyde activation of polymer Nylon-6 for lipase immobilization: enzyme characteristics and stability.

An extracellular alkaline lipase of a thermo tolerant Bacillus coagulans BTS-3 was immobilized onto glutaraldehyde activated Nylon-6 by covalent binding. Under optimum conditions, the immobilization yielded a protein loading of 228 microg/g of Nylon-6. Immobilized enzyme showed maximum activity at a temperature of 55 degrees C and pH 7.5. The enzyme was stable between pH 7.5-9.5. It retained 88% of its original activity at 55 degrees C for 2h and also retained 85% of its original activity after eight cycles of hydrolysis of p-NPP. Kinetic parameters Km and Vmax were found to be 4mM and 10 micromol/min/ml, respectively. The influence of organic solvents on the catalytic activity of immobilized enzyme was also evaluated. The bound lipase showed enhanced activity when exposed to n-heptane. The substrate specificity of immobilized enzyme revealed more efficient hydrolysis of higher carbon length (C-16) ester than other ones.

Pharmacological and clinical evaluation of L-asparaginase in the treatment of leukemia.

L-Asparaginase is an effective antineoplastic agent, used in the acute lymphoblastic leukemia chemotherapy. It has been an integral part of combination chemotherapy protocols of pediatric acute lymphoblastic leukemia for almost 3 decades. The potential of L-asparaginase as a drug of leukemia has been a matter of discussion due to the high rate of allergic reactions exhibited by the patients receiving the medication of this enzyme drug. Frequent need of intramuscular injection has been another disadvantage associated with the native preparation. However, of late these clinical complications seem to have been addressed by modified versions of L-asparaginase. PEG-L-asparaginase proves to be most effective in this regard. It becomes important to discuss the efficacy of L-asparaginase as an antileukemic drug vis-a-vis these disadvantages. In this review, an attempt has been made to critically evaluate the pharmacological and clinical potential of various preparations of L-asparaginase as a drug. Advantages of PEG-L-asparaginase over native preparations and historical developments of therapy with l-asparaginase have also been outlined in the review below.

Production, purification, and characterization of lipase from thermophilic and alkaliphilic Bacillus coagulans BTS-3.

A thermophilic isolate Bacillus coagulans BTS-3 produced an extracellular alkaline lipase, the production of which was substantially enhanced when the type of carbon source, nitrogen source, and the initial pH of culture medium were consecutively optimized. Lipase activity 1.16 U/ml of culture medium was obtained in 48 h at 55 degrees C and pH 8.5 with refined mustard oil as carbon source and a combination of peptone and yeast extract (1:1) as nitrogen sources. The enzyme was purified 40-fold to homogeneity by ammonium sulfate precipitation and DEAE-Sepharose column chromatography. Its molecular weight was 31 kDa on SDS-PAGE. The enzyme showed maximum activity at 55 degrees C and pH 8.5, and was stable between pH 8.0 and 10.5 and at temperatures up to 70 degrees C. The enzyme was found to be inhibited by Al3+, Co2+, Mn2+, and Zn2+ ions while K+, Fe3+, Hg2+, and Mg2+ ions enhanced the enzyme activity; Na+ ions have no effect on enzyme activity. The purified lipase showed a variable specificity/hydrolytic activity towards various 4-nitrophenyl esters.