Vilas Shukla, Ph.D, PMP

https://www.biophorum.com/download/a-risk-based-approach-to-filter-integrity-testing-requirements-for-biologics-drug-substance-manufacturers/

Escherichia coli W3110 derivatives, strains SZ63 and SZ85, were previously engineered to produce optically pure d(−) and l( )-lactate from hexose and pentose sugars. To expand the substrate range, a cluster of sucrose genes (cscR′ cscA cscKB) was cloned and characterized from E. coli KO11. The resulting plasmid was functionally expressed in SZ63 but was unstable in SZ85. Over 500 mm d(−)-lactate was produced from sucrose and from molasses by SZ63(pLOI3501).

Comparison of the production of L-phenylacetylcarbinol (L-PAC) from benzaldehyde by cells
of Torulaspora delbrueckii immobilized in calcium alginate and barium alginate showed that cells
immobilized in calcium alginate beads were usable for up to six cycles compared with nine cycles for barium alginate-immobilized cells. Barium alginate-immobilized cells yielded a total of 2.17 g of L-PAC in nine cycles whereas in the case of calcium alginate-immobilized cells, a total of 1.17 g of L-PAC… Show more

Comparison of the production of L-phenylacetylcarbinol (L-PAC) from benzaldehyde by cells
of Torulaspora delbrueckii immobilized in calcium alginate and barium alginate showed that cells
immobilized in calcium alginate beads were usable for up to six cycles compared with nine cycles for barium alginate-immobilized cells. Barium alginate-immobilized cells yielded a total of 2.17 g of L-PAC in nine cycles whereas in the case of calcium alginate-immobilized cells, a total of 1.17 g of L-PAC was formed when 400mg benzaldehyde and 400mm3 of acetaldehyde were used in each cycle as substrate and co-substrate respectively. Show less

In a 5 dm3 stirred tank reactor, bioconversion of 30 g benzaldehyde by cells of Torulaspora delbrueckii yielded 22.9 g of pure l-phenylacetylcarbinol (l-PAC). Facile functional group transformation of 4.5 g of l-PAC to 2-(methylimino)-1-phenyl-1-propanol by exposure to microwave irradiation for 9 min resulted in 2.48 g of product. Conversion of 4.8 g of 2-(methylimino)-1-phenyl-1-propanol to 3.11 g of ephedrine was achieved by exposure to microwaves in a reaction time of 10 min. The identity of… Show more

In a 5 dm3 stirred tank reactor, bioconversion of 30 g benzaldehyde by cells of Torulaspora delbrueckii yielded 22.9 g of pure l-phenylacetylcarbinol (l-PAC). Facile functional group transformation of 4.5 g of l-PAC to 2-(methylimino)-1-phenyl-1-propanol by exposure to microwave irradiation for 9 min resulted in 2.48 g of product. Conversion of 4.8 g of 2-(methylimino)-1-phenyl-1-propanol to 3.11 g of ephedrine was achieved by exposure to microwaves in a reaction time of 10 min. The identity of all the products was confirmed by 1H NMR and FT-IR analysis. Show less

Studies were carried out to explore the possibility of decreasing the toxic and inhibitory effects of the substrate benzaldehyde during its biotransformation to L-PAC by free cells of Torulaspora delbrueckii using b -cyclodextrin (b -CD). Use of various levels of benzaldehyde and acetaldehyde in presence of 2% of b -CD showed that, in presence of b -CD, the organism could tolerate higher levels of benzaldehyde and acetaldehyde. Semi-continuous feeding of benzaldehyde and acetaldehyde was found… Show more

Studies were carried out to explore the possibility of decreasing the toxic and inhibitory effects of the substrate benzaldehyde during its biotransformation to L-PAC by free cells of Torulaspora delbrueckii using b -cyclodextrin (b -CD). Use of various levels of benzaldehyde and acetaldehyde in presence of 2% of b -CD showed that, in presence of b -CD, the organism could tolerate higher levels of benzaldehyde and acetaldehyde. Semi-continuous feeding of benzaldehyde and acetaldehyde was found to increase the yield of L-PAC in comparison with one time feeding. Show less

The effect of process parameters on the biotransformation of benzaldehyde to L-phenylacetylcarbinol (L-PAC) using a yeast isolate identified as Torulaspora delbrueckii was studied. The maximum yield of L-PAC obtained was (331 mg) per 100 ml biotransformation medium (glucose 3%, peptone 0.6% and at pH 4.5) from 600 mg of benzaldehyde with 8 h of reaction at 30 ± 2 °C. Growing the organism in presence of 3% glucose reduced the biotransformation time to 120 min. Addition of 0.6% acetaldehyde… Show more

The effect of process parameters on the biotransformation of benzaldehyde to L-phenylacetylcarbinol (L-PAC) using a yeast isolate identified as Torulaspora delbrueckii was studied. The maximum yield of L-PAC obtained was (331 mg) per 100 ml biotransformation medium (glucose 3%, peptone 0.6% and at pH 4.5) from 600 mg of benzaldehyde with 8 h of reaction at 30 ± 2 °C. Growing the organism in presence of 3% glucose reduced the biotransformation time to 120 min. Addition of 0.6% acetaldehyde (30–35%) lead to an increase in L-PAC yield to 450 mg%. Semi-continuous feeding of benzaldehyde (200 mg) and acetaldehyde (200 μl) four times at 30 min intervals could produce 683 mg of L-PAC/100 ml biotransformation medium. Chiral HPLC analysis of purified L-PAC and PAC-diol showed 99% enantiomeric purity. The cell mass was found to be reusable for biotransformation up to nine times when benzaldehyde and acetaldehyde levels were maintained at (350 mg and 350 μl)–(400 mg and 400 μl). At concentrations from 450 mg and 450 μl to 600 mg and 600 μl, however the cell mass could give efficient biotransformation only during one use. Show less

The gas–liquid mass transfer coefficient KLa in the fermenter is a strong function of mode of energy dissipation and physico-chemical properties of the liquid media. A combination of disc turbine (DT) and pitched blade turbine down flow (PTD) impellers has been tested in laboratory bioreactor for gas hold-up and gas–liquid mass transfer performance for the growth and biotransformation medium for an yeast isolate VS1 capable of biotransforming benzaldehyde to l-phenyl acetyl carbinol (l-PAC) and… Show more

The gas–liquid mass transfer coefficient KLa in the fermenter is a strong function of mode of energy dissipation and physico-chemical properties of the liquid media. A combination of disc turbine (DT) and pitched blade turbine down flow (PTD) impellers has been tested in laboratory bioreactor for gas hold-up and gas–liquid mass transfer performance for the growth and biotransformation medium for an yeast isolate VS1 capable of biotransforming benzaldehyde to l-phenyl acetyl carbinol (l-PAC) and compared with those in water.

Correlations have been developed for the prediction of the fractional gas hold-up and gas–liquid mass transfer coefficient for the above media. The mass transfer coefficient and respiration rate have been determined in the shake flask for the growth as well as for biotransformation medium. These results, then have been used to optimize the operating parameters (impeller speed and aeration) for growth and biotransformation in a laboratory bioreactor. The comparison of cell mass production and l-PAC production in the bioreactor has been done with that obtained in shake flask studies. Show less

Fermentative production of squalene under anaerobic conditions using commercially available compressed baker's yeast (Saccharomyces cerevisiae), and a strain of Torulaspora delbrueckii isolated from molasses was studied. Yield of squalene from S. cerevisiae and T. delbrueckii were found to be 41.16 and 237.25 μg g−1 respectively, dry weight of yeast cells. Isolation and purification of squalene from the lipid extracts obtained by cell lysis of either strain were achieved chromatographically… Show more

Fermentative production of squalene under anaerobic conditions using commercially available compressed baker's yeast (Saccharomyces cerevisiae), and a strain of Torulaspora delbrueckii isolated from molasses was studied. Yield of squalene from S. cerevisiae and T. delbrueckii were found to be 41.16 and 237.25 μg g−1 respectively, dry weight of yeast cells. Isolation and purification of squalene from the lipid extracts obtained by cell lysis of either strain were achieved chromatographically. The purified squalene was characterized spectroscopically against an authentic standard. Show less

The objective of the present work was to study the stereoselective synthesis of homo-allylic alcohol using stereoselective hydrolysis by Rhizopus arrhizus and also to study the stereoselective synthesis of allyl phenyl carbinol (APC) or 1-Phenyl-3-butene-1-o by combination of chemical synthesis and biotransformation from cheap raw materials such as benzaldehyde and allyl bromide. Stereospecific synthesis of allyl phenyl carbinol (APC) was achieved by acetate hydrolysis by R. arrhizus giving R… Show more

The objective of the present work was to study the stereoselective synthesis of homo-allylic alcohol using stereoselective hydrolysis by Rhizopus arrhizus and also to study the stereoselective synthesis of allyl phenyl carbinol (APC) or 1-Phenyl-3-butene-1-o by combination of chemical synthesis and biotransformation from cheap raw materials such as benzaldehyde and allyl bromide. Stereospecific synthesis of allyl phenyl carbinol (APC) was achieved by acetate hydrolysis by R. arrhizus giving R ( ) enantiomer. Maximum enantiomeric excess of APC was obtained at 16 h where as maximum yield of it was obtained at 48 h of biotransformation. Show less

L-Phenylacetylcarbinol (L-PAC), an important drug intermediate, can be produced by biotransformation of benzaldehyde, mainly by yeast cultures but also by Zymomonas mobilis. The biotransformation by free cells, immobilized cells, mutant organisms, purified pyruvate decarboxylase as well as the use of bioreactors, the downstream processing of L-PAC and the industrial applications have been reviewed.

Ether extract from the biotransformation broth from bezaldehyde to L-PAC conversion using compressed baker’s yeast was subjected to column chromatography using silica gel. The presence of L-PAC and PAC-diol in the eluted fractions were confirmed on the basis of sprectal analysis. An easy method of down stream processing of L-PAC based on bisulphite adduct preparation was developed.