Rajinder Parshad

Immobilization of Lipase by Entrapment in Ca-alginate Beads

The lipase-producing strain, Arthrobacter sp. (ABL), isolated was immobilized in Ca-alginate beads by entrapment. The alginate beads were prepared as an aqueous mixture of sodium alginate, the cells and CaCl2 to increase its reusability, and overall enzyme stability. Various parameters like alginate and CaCl2 concentration, lipase units loading and bead size were evaluated for optimum immobilization yield. It was observed that with the increase in alginate concentration, the yield of immobilized enzyme also increased up to a limit. A similar pattern was observed with CaCl2 addition; the optimum concentrations of alginate and CaCl2 observed were 1.5% (w/v) and 2%, respectively. The concentration of enzyme entrapped in the beads with an activity of 5 units per gram of wet beads was obtained by the addition of 100 units in 10 mL of slurry; beyond this amount a very little increase in activity was observed. The maximum immobilization yield was observed with a 1.2 mm bead size; increased bead sizes decreased the yield of immobilization. After optimization of all the parameters, a 40% yield of lipase (ABL) activity was observed in the Ca-alginate beads. These lipase beads were used for 10 cycles for the hydrolysis of triglycerides without any loss in activity. The entrapped lipase was more stable over a wide range of temperatures, pH, and storage time as compared to free enzyme.

Purification and characterisation of an ester hydrolase from a strain of Arthrobacter species: Its application in asymmetrisation of 2-benzyl-1,3-propanediol acylates☆

An ester hydrolase (ABL) has been isolated from a strain of Arthrobacter species (RRLJ-1/95) maintained in the culture collection of this laboratory. The purified enzyme has a specific activity of 1700 U/mg protein and is found to be composed of a single subunit (Mr 32,000), exhibiting both lipase and esterase activities shown by hydrolysis of triglycerides and p-nitrophenyl acetate respectively. Potential application of the enzyme concerns the asymmetrisation of prochiral 2-benzyl-1,3-propanediol esters besides enantioselective hydrolysis of alkyl esters of unsubstituted and substituted 1-phenyl ethanols.

Enzymatic resolution of naproxen

Abstract Trichosporon sp. (TSL), a newly found strain isolated from a locally fermented cottage cheese has been found to be highly stereoselective in the resolution of ( S )-(+)-naproxen (ee >99%, E ∼500) from the corresponding racemic methyl ester. The process of resolution using whole cells has been scaled up to multi-kg level. Optimization of experimental conditions including downstream processing at 80–100 g/L substrate concentration with >90% recovery has been achieved. Changes in the physical parameters such as the particle size of the substrate play an important role in the resolution kinetics. A new strain of Trichosporon sp. having high cell density in cultivation (>60 g dry cell mass L −1 in 14–16 h) is found to be sufficiently stable for two years in dry powder form at 5–8°C. The viability of the resolution process has been further improved by the development of a simple racemization process for the enriched ( R )-(−)-ester.

Enantioselective hydrolysis of alkyl esters of substituted 1-phenyl ethanols using newly isolated strain of Arthrobacter sp.; a comparative study with known commercial lipases

Abstract The enantioselective hydrolysis of alkyl esters of substituted 1-phenyl ethanol racemates has been conducted using a newly selected strain of Arthrobacter sp. (ABL) and its efficacy compared with commercially available enzymes such as PPL (porcine pancreatic lipase), CCL (Candida cylindracea) and PSL (Pseudomonas sp.). The ABL has demonstrated reasonable superiority over the above three known enzymes both in terms of its enantioselectivity as well as the overall rate of hydrolysis.

Application of a statistically enhanced, novel, organic solvent stable lipase from Bacillus safensis DVL-43

This paper presents the molecular identification of a newly isolated bacterial strain producing a novel and organic solvent stable lipase, statistical optimization of fermentation medium, and its application in the synthesis of ethyl laurate. On the basis of nucleotide homology and phylogenetic analysis of 16S rDNA sequence, the strain was identified as Bacillus safensis DVL-43 (Gen-bank accession number KC156603). Optimization of fermentation medium using Plackett-Burman design and response surface methodology led to 11.4-fold increase in lipase production. The lipase from B. safensis DVL-43 exhibited excellent stability in various organic solvents. The enzyme retained 100% activity after 24h incubation in xylene, DMSO and toluene, each solvent being used at a concentration of 25% (v/v). The use of partially purified DVL-43 lipase as catalyst in the synthesis of ethyl laurate, an esterification product of lauric acid and ethanol, resulted in 80% esterification in 12h under optimized conditions. The formation of ethyl laurate was confirmed using TLC and (1)H NMR. Organic solvent stable lipases exhibiting potential application in enzymatic esterification are in great demand in flavor, fine chemicals and pharma industries. We could not find any report on lipase production from B. safensis strain and its application in esterification.

Arthrobacter sp. lipase immobilization for preparation of enantiopure masked β-amino alcohols

Recent reports on immobilization of lipase from Arthrobacter sp. (ABL, MTCC 5125; IIIM isolate) on insoluble polymers have shown altered properties including stability and enantioselectivity. Present work demonstrates a facile method for the preparation of enantiopure beta-amino alcohols by modulation of ABL enzyme properties via immobilization on insoluble as well as soluble supports using entrapment/covalent binding techniques. Efficacies of immobilized ABL on insoluble supports prepared from tetraethylorthosilicate/aminopropyltriethoxy silane and soluble supports derived from copolymerization of N-vinyl pyrrolidone-allylglycidyl ether (ANP type)/N-vinyl pyrrolidone-glycidyl methacrylate (GNP type) for kinetic resolution of masked beta-amino alcohols have been studied vis-à-vis free ABL enzyme/wet cell biomass. The immobilized lipase on different insoluble/soluble supports has shown 21-110 mg/g protein binding and 30-700 U/g activity for hydrolyzing tributyrin substrate. The findings have shown a significant enhancement in enantioselectivity (ee 99%) vis-à-vis wet cell biomass providing ee 70-90% for resolution of beta-amino alcohols.

Purification and characterization of a novel enantioselective hydrolase from Bacillus subtilis.

Screening of the micro‐organisms from an in‐house microbial culture repository, identified a bacterial strain bearing membrane‐bound, inducible ester hydrolase activity. The strain designated as RRL‐BB1 has been identified as Bacillus subtilis by 16 S rRNA typing. Its application in the kinetic resolution of several racemates, including drug intermediates, showed moderate to high enantioselectivity. The enzyme, designated as BBL, exhibited high enantioselectivity (ee ≈99%) with acyl derivatives of unsubstituted and substituted 1‐(phenyl)ethanols and 1‐(6‐methoxy‐2‐naphthyl)ethanols. With acyl derivatives of 2‐(6‐methoxy‐2‐naphthyl)propan‐1‐ol, moderate enantioselectivity was observed. The enzyme also showed moderate enantioselectivity with alkyl esters of carboxylic acids i.e. 2‐bromopropanoic acid and 2‐hydroxy‐4‐phenylbutanoic acid. The enzyme was purified to >90% purity from cell‐free extract of RRL‐BB1 with 26% overall yield. The purified enzyme exhibited hydrolase activity without any noticeable decrease in the rate of hydrolysis or the enantioselectivity profile. A specific activity of 450 units/mg protein resulted after at least a 200‐fold purification of the crude cell‐free extract. The key purification step was the irreversible adsorption of the salt‐precipitated crude enzyme on hydrophobic resin, in the presence of a low salt concentration, and desorption of the enzyme with a linear gradient of 1% sodium cholate. The purified enzyme was a 45 kD monomer as shown by SDS/PAGE. The N‐terminal amino acid sequence of the purified enzyme was determined as Thr‐Lys‐Leu‐Thr‐Val‐Gln‐Thr‐Arg‐Asp‐Gly‐Ala‐Leu‐Arg‐Gly‐Thr. The N‐terminal sequence did not bear any homology with other known bacterial lipases. BBL is maximally active at 37 °C, pH 8.0 and fairly stable up to 40 °C, pH 6–10. The enzyme is insensitive to EDTA but inhibited by serine protease inhibitor PMSF. Its activity (72%) was retained in the presence of the anionic detergent SDS at a concentration of 0.2% (w/v).

A facile approach towards enantiomerically pure masked β-amino alcohols

β-Amino alcohols are bioactive molecules, used also as catalysts in asymmetric C–C bond formation. While asymmetric synthesis has been the preferred route for their preparation, there was always been a need to develop a facile methodology involving environmentally friendly transformations. Masked amines in the form of phthalimide alcohols, prepared via a fast coupling reaction in an ionic liquid as a reusable reaction media together with reduction and an efficient biocatalytic resolution, offer a green methodology for enantiomerically pure products (ee > 99%, 50 g L−1).

Macroporous Beads for Lipase Immobilization Kinetic Resolution of a Racemic Drug Intermediate

Lipase isolated from Arthrobacter sp. (RRLJ-1, MTCC No. 5125, named ABL), is effective in resolving a wide range of racemic drug intermediates. In this study, ABL was immobilized on a series of synthetic macroporous epoxy copolymers beads with varying pore sizes, surface area and hydrophobicity. Poly(glycidyl methacrylate-co-ethylene dimethacrylate) beads, with 75% crosslink density and 10% of epoxy groups modified with dibutyl amine [GMA-EGDM-75 (10% DBA)] had a pore volume of 0.77 mL/g and a surface area of 86.05 m 2/g; these beads were optimally suitable for ABL immobilization. The covalent binding of the lipase was optimized by varying the ionic strength, buffers, pH, temperature and time. The optimal binding was achieved in 100 mM phosphate buffer at 4°C, pH 7.0 in three hours. Under these conditions the polymer retained 34 units and 12 mg of ABL per gram. Immobilized ABL displayed enhanced thermal, organic solvent and pH stability compared to the free enzyme. The immobilized enzyme was used repeatedly (fifteen cycles) to resolve the fluoxitine intermediate (racemic ethyl-3-hydroxy-3-phenyl propanoate) without any loss in stereospecificity. The resolution time of fluoxitine intermediate was reduced to almost half (from 84 to 48 hours) by using the immobilized enzyme.

Chrysomycins A–C, antileukemic naphthocoumarins from Streptomyces sporoverrucosus

Two known naphthocoumarins, chrysomycins A (1) and B (2), along with one new naphthocoumarin chrysomycin C (3) were isolated from the antimicrobial strain of Streptomyces sporoverrucosus (MTCC11715) (isolated from soil samples from the Jammu hills) and characterized. The structure of the new compound 3 was established from 2D-NMR data. Chrysomycins A (1) and B (2) were identified using a strategic HPLC–PDA/LCMS and Dictionary of Natural Products (DNP) based fast dereplication. Additionally, two new naphthocoumarins, chrysomycins D and E were identified using LCMS, UV and DNP information. Chrysomycins A–C (1–3) were isolated for the first time from Streptomyces sporoverrucosus and were screened for cytotoxicity against a panel of cancer cell lines (A549, Colo205, PC-3, MIAPaCa-2, and HL-60), amongst which the most potent activity was observed against human leukemia HL-60 cells with IC50 values of 0.9, 0.95 and 11 μM, respectively. The mechanistic studies indicated that chrysomycins A (1) and B (2), at 1 μM concentration, distorted the cellular and nuclear morphology with significant DNA damage and apoptosis in HL-60 cells.