R.K. Saxena

Characterization and wash performance analysis of an SDS-stable alkaline protease from a Bacillus sp.

An alkaline, SDS-stable protease optimally active at pH 11 from a Bacillus sp. RGR-14 was produced in a complex medium containing soybean meal, starch and calcium carbonate. The protease was active over a wide temperature range of 20–80 °C with major activity between 45 and 70 °C. The protease was completely stable for 1 h in 0.1% SDS and retained 70% of its activity in the presence of 0.5% SDS after 1 h of incubation. The enzyme was active in presence of surfactants (ionic and non-ionic) with 29% enhancement in activity in Tween-85 and was also stable in various oxidizing agents with 100 and 60% activity in presence of 1% sodium perborate and 1% H2O2, respectively. The enzyme was also compatible with commercial detergents (1% w/v) such as Surf, Ariel, Wheel, Fena and Nirma, retaining more than 70% activity in all the detergents after 1 h. Wash performance analysis of grass and blood stains on cotton fabric showed an increase in reflectance (14 and 25% with grass and blood stains, respectively) after enzyme treatment. However, enzyme in conjunction with detergent proved best, with a maximum reflectance change of 46 and 34% for grass and blood stain removal, respectively, at 45 °C. Stain removal was also effective after protease treatment at 25 and 60 °C.

Microbial production and applications of 1,2-propanediol

Abstract1,2-Propanediol (propylene glycol) is an existing commodity chemical and can be produced from renewable resources using microbes. By virtue of being a natural product, relevant biochemical pathways can be harnessed into fermentation processes to produce 1,2-propanediol. In the present review, the chemical process and different biological strategies for the production of 1,2-propanediol are reviewed and compared with the potentials and limitations of all processes. For the successful commercial production of this diol, it is necessary to establish the metabolic pathways and production hosts (microorganisms), which are capable of delivering final product with high yields and volumetric productivity. Three pathways which have been recognized for 1,2-propanediol production are discussed here. In the first, de-oxy sugars like fucose and rhamnose are used as the carbon sources, while in the other route, the glycolytic intermediate-dihydroxyacetonephosphate (DHAP) is used to produce 1,2-propanediol via the formation of methylglyoxal. A new pathway of 1,2-propanediol production by lactic acid degradation under anoxic conditions and the enzymes involved is also discussed. The production of this diol has gained attention because of their newer applications in industries such as polymers, food, pharmaceuticals, textiles, etc. Furthermore, improvement in fermentation technology will permit its uses in other applications. Future prospect in the light of the current research and its potential as a major bulk chemical are discussed.

A hyper-thermostable, alkaline lipase from Pseudomonas sp. with the property of thermal activation

A hyper-thermostable, alkaline lipase from a newly-isolated, mesophilic Pseudomonas sp. was optimal at pH 11 and at 90 °C. It had a half-life of more than 13 h at 90 °C. It was activated by 30% when heated at 90 °C for 2 h. The enzyme had a greater affinity for mustard oil (Km=40 mg ml−1) than for olive oil (Km=140 mg ml−1).

Efficient production of L-asparaginase from Bacillus licheniformis with low-glutaminase activity: optimization, scale up and acrylamide degradation studies.

L-Asparaginase has potential as an anti-cancer drug and for prevention of acrylamide formation in fried and baked foods. Production of the enzyme by Bacillus licheniformis (RAM-8) was optimized by process engineering using a statistical modeling approach and a maximum yield of 32.26 IU/ml was achieved. The L-asparaginase exhibited glutaminase activity of only 0.8 IU/ml and would therefore be less prone to cause the side effects associated with asparaginase therapy compared to enzyme preparations with higher glutaminase activities. When production was carried out in a 30-L bioreactor, enzyme production reached 29.94 IU/ml in 15 h. The enzyme inhibited poly-acrylamide formation in 10% acrylamide solution and reduced acrylamide formation in fried potatoes by 80%.

Characterization of cross-linked immobilized lipase from thermophilic mould Thermomyces lanuginosa using glutaraldehyde.

Cross-linked enzyme aggregates (CLEAs) have emerged as an interesting biocatalyst design for immobilization. Using this approach, a 1,3 regiospecific, alkaline and thermostable lipase from Thermomyces lanuginosa was immobilized. Efficient cross-linking was observed when ammonium sulphate was used as precipitant along with a two fold increase in activity in presence of SDS. The TEM and SEM microphotographs of the CLEAs formed reveal that the enzyme aggregates are larger in size as compared to the free lipase due to the cross-linking of enzyme aggregates with glutaraldehyde. The stability and reusability of the CLEA with respect to olive oil hydrolysis was evaluated. The CLEA showed more than 90% residual activity even after 10 cycles of repeated use.

Fermentation and downstream processing of lipase from Aspergillus terreus

Fermentation behaviour of Aspergillus terreus lipase was studied in a 10 l fermentor. Lipase production was enhanced to 14 200 U l−1 in 54 h in the fermentor as against 7000 U l−1 in 96 h in shake flasks under optimised nutritional conditions. A 2.4-fold increase in specific activity (16.2 U mg−1 protein) was also attained. Inoculum density, dissolved oxygen levels and agitation were the major controlling factors. A two-step cost-effective downstream processing methodology comprising of an aqueous two-phase system (ATPS) of polyethylene glycol (PEG) and phosphate was devised. This procedure resulted in a 12-fold purification of the lipase with 100% yield in less than 1 h.

Degradation of alpha, beta, gamma and delta-hexachlorocyclohexanes by Sphingomonas paucimobilis

Sphingomonas paucimobilis degrades aerobically α, β, γ and δ-hexachlorocyclohexane. With α-HCH, complete degradation occurred after 3 days but with β and γ, and with δ-HCH, 98 and 56 % degradation occurred after 12 and 8 days of incubation, respectively. Pentachlorocyclohexene was formed as the primary metabolite during the degradation of all the HCH isomers.

Microwave-assisted rapid characterization of lipase selectivities

A rapid screening procedure for characterization of lipase selectivities using microwaves was developed. The rate of reaction of various commercial lipases (porcine pancreas, Mucor miehei, Candida rugosa, Pseudomonas cepacia) as well as lipases from laboratory isolates-Bacillus stearothermophilus and Burkholderia cepacia RGP-10 for triolein hydrolysis was 7- to 12-fold higher in a microwave oven as compared to that by pH stat. The esterification of sucrose/methanol and ascorbic acid with different fatty acids was also achieved within 30 s in a microwave using porcine pancreas, B. stearothermophilus SB-1 and B. cepacia RGP-10 lipases. The relative rates and selectivity of the lipases both for hydrolytic and synthesis reactions remains unaltered. However, the rate of reaction was dynamically enhanced when exposed to microwaves. Microwave-assisted enzyme catalysis can become an attractive procedure for rapid characterization of large number of enzyme samples and substrates, which otherwise is a cumbersome and time-consuming exercise.

Evaluation of corncob hemicellulosic hydrolysate for xylitol production by adapted strain of Candida tropicalis.

A maximum xylose extraction of 21.98 g/L was obtained in hydrolysate with a solid to liquid ratio of 1:8 (w/v) at 1% H(2)SO(4) and treated for 30 min. The optimized and treated corncob hemicellulosic hydrolysate medium supplemented with (g/L) yeast extract 5.0, KH(2)PO(4) 2.0, MgSO(4)·7H(2)O 0.3 and methanol 10 mL whose pH was adjusted to 4.5 acts as production medium. Under this condition; the adapted strain of C. tropicalis resulted in 1.22-fold increase in xylitol yield and 1.70-fold enhancement in volumetric productivity was obtained as compared to parent strain of C. tropicalis. On concentrating the hydrolysate under vacuum using rotavapor proves to be efficient in terms of improved xylitol yield and productivity over microwave assisted concentration using adapted strain of C. tropicalis. The immobilized cells of C. tropicalis resulted in more than 70% efficiency up to third cycle. The xylitol production could be scaled up to 10 L fermentor.

Expanding horizons of shikimic acid

Shikimic acid is an industrially important chiral compound used as a key ingredient in formulation of drug Oseltamivir phosphate (Tamiflu) for the treatment of swine/avian flu. The high cost and limited availability of shikimic acid isolated from plants has detained the use of this valuable building block of the drug. It is a versatile compound having many characteristic properties for many synthetic reactions particularly in pharmaceuticals and cosmetic industries. By virtue of being a natural product, the relevant biochemical pathway in microorganisms can be harnessed into fermentation processes to produce shikimic acid. This is an excellent alternative for the sustainable and efficient production of shikimic acid over the tedious and cumbersome process of plant based extraction methods. Various strategies of shikimic acid production are reviewed and an account of comparison of their challenges, promises and restraint is presented. Furthermore, present review attempts to focus on the market trend of shikimic acid due to its high demand with particular emphasis laid on the pandemics of swine flu. This review not only covers the recent advances in shikimic acid production but also highlights the versatile applications and its market scenario. The concluding remarks and its potential as a commercial bulk chemical are discussed in the light of current research.