Polylactic acid: synthesis and biomedical applications.

Social and economic development has driven considerable scientific and engineering efforts on the discovery, development and utilization of polymers. Polylactic acid (PLA) is one of the most promising biopolymers as it can be produced from nontoxic renewable feedstock. PLA has emerged as an important polymeric material for biomedical applications on account of its properties such as biocompatibility, biodegradability, mechanical strength and process ability. Lactic acid (LA) can be obtained by fermentation of sugars derived from renewable resources such as corn and sugarcane. PLA is thus an eco-friendly nontoxic polymer with features that permit use in the human body. Although PLA has a wide spectrum of applications, there are certain limitations such as slow degradation rate, hydrophobicity and low impact toughness associated with its use. Blending PLA with other polymers offers convenient options to improve associated properties or to generate novel PLA polymers/blends for target applications. A variety of PLA blends have been explored for various biomedical applications such as drug delivery, implants, sutures and tissue engineering. PLA and their copolymers are becoming widely used in tissue engineering for function restoration of impaired tissues due to their excellent biocompatibility and mechanical properties. The relationship between PLA material properties, manufacturing processes and development of products with desirable characteristics is described in this article. LA production, PLA synthesis and their applications in the biomedical field are also discussed.

Differential induction, purification and characterization of cold active lipase from Yarrowia lipolytica NCIM 3639.

The production, purification and characterization of cold active lipases by Yarrowia lipolytica NCIM 3639 is described. The study presents a new finding of production of cell bound and extracellular lipase activities depending upon the substrate used for growth. The strain produced cell bound and extracellular lipase activity when grown on olive oil and Tween 80, respectively. The organism grew profusely at 20 °C and at initial pH of 5.5, producing maximum extracellular lipase. The purified lipase has a molecular mass of 400 kDa having 20 subunits forming a multimeric native protein. Further the enzyme displayed an optimum pH of 5.0 and optimum temperature of 25 °C. Peptide mass finger printing reveled that some peptides showed homologues sequence (42%) to Yarrowia lipolytica LIP8p. The studies on hydrolysis of racemic lavandulyl acetate revealed that extracellular and cell bound lipases show preference over the opposite antipodes of irregular monoterpene, lavandulyl acetate.

Comparative production of cellulases by mutants of Penicillium janthinellum NCIM 1171 and its application in hydrolysis of Avicel and cellulose.

Mutants of Penicillium janthinellum NCIM 1171 were evaluated for cellulase production using both submerged fermentation (SmF) and solid state fermentation (SSF). Mutant EU2D-21 gave highest yields of cellulases in both SmF and SSF. Hydrolysis of Avicel and cellulose were compared using SmF and SSF derived enzyme preparations obtained from EU2D-21. Surprisingly, the use of SSF derived preparation gave less hydrolysis compared to SmF derived enzymes. This may be due to inactivation of ß-glucosidase at 50°C in SSF derived enzyme preparations. SmF derived enzyme preparations contained both thermostable and thermosensitive ß-glucosidases where as SSF derived enzyme preparations contained predominantly thermosensitive ß-glucosidase. This is the first report on less thermostability of SSF derived ß-glucosidase which is the main reason for getting less hydrolysis.

Development of biocatalysts for production of commodity chemicals from lignocellulosic biomass.

Lignocellulosic biomass is recognized as potential sustainable source for production of power, biofuels and variety of commodity chemicals which would potentially add economic value to biomass. Recalcitrance nature of biomass is largely responsible for the high cost of its conversion. Therefore, it is necessary to introduce some cost effective pretreatment processes to make the biomass polysaccharides easily amenable to enzymatic attack to release mixed fermentable sugars. Advancement in systemic biology can provide new tools for the development of such biocatalysts for sustainable production of commodity chemicals from biomass. Integration of functional genomics and system biology approaches may generate efficient microbial systems with new metabolic routes for production of commodity chemicals. This paper provides an overview of the challenges that are faced by the processes converting lignocellulosic biomass to commodity chemicals. The critical factors involved in engineering new microbial biocatalysts are also discussed with more emphasis on commodity chemicals.

Strain improvement of Lactobacillus lactis for D-lactic acid production.

Three mutants, isolated by repeated UV mutagenesis of Lactobacillus lactis NCIM 2368, produced increased D: -lactic acid concentrations. These mutants were compared with the wild type using 100 g hydrolyzed cane sugar/l in the fermentation medium. One mutant, RM2-24, produced 81 g lactic acid/l which was over three times that of the wild type. The highest D: -lactic acid (110 g/l) in batch fermentation was obtained with 150 g cane sugar/l with a 73% lactic acid yield. The mutant utilizes cellobiose efficiently, converting it into D-lactic acid suggesting the presence of cellobiase. Thus, this strain could be used to obtain D-lactic acid from cellulosic materials that are pre-hydrolyzed with cellulase.

Purification and characterization of acidic lipase from Aspergillus niger NCIM 1207.

An extracellular lipase from Aspergillus niger NCIM 1207 has been purified to homogeneity using ammonium sulfate precipitation followed by phenyl sepharose and Sephacryl-100 gel chromatography. This protocol resulted in 149 fold purification with 54% final recovery. The purified enzyme showed a prominent single band on SDS-PAGE. The purified enzyme is a monomeric protein of 32.2 kDa molecular weight and exhibits optimal activity at 50 degrees C. One interesting feature of this enzyme is its highly acidic pH optimum. The isoelectric point (pI) of lipase was 8.5. The purified lipase appears to be unique since it cleaved triolein at only 3-position releasing 1,2-diolein. Chemical modification studies revealed that His, Ser, Carboxylate and Trp are involved in catalysis.

Environment friendly crosslinked chitosan as a matrix for selective adsorption and purification of lipase of Aspergillus niger.

Chitosan and its derivatives have been used as affinity matrices for purification of lipase from Aspergillus niger NCIM 1207. Trimellitic anhydride (TMA)-crosslinked deacetylated chitin adsorbed lipase selectively, yielding approximately 5-fold purification of the crude lipase with 70% yield. Further 9-fold purification occurred on eluting through Sephacryl-100. These results suggest that chitosan derivatives can be used as inexpensive biopolymer matrices for the purification of lipases for industrial applications.

Metal complexes of crosslinked chitosans Part II. An investigation of their hydrolysis to chitooligosaccharides using chitosanase.

This paper investigates the behavior of crosslinked chitosans and metal-complexed crosslinked chitosans under similar hydrolytic conditions. Crosslinked chitosans with trimellitic anhydride, diisocyanatohexane, and dibromodecane as crosslinking agents under heterogenous reaction conditions were used as metal complexing agents by equilibrating them with metal salts such as ZnCl(2), MnSO(4), CuSO(4), CdSO(4), Pb(NO(3))(2), and HgCl(2). Crosslinked chitosan without metal complexation had the same hydrolytic behavior as uncrosslinked chitosan. However, when the crosslinked chitosans were complexed with metals, their rates of hydrolysis and extent of hydrolysis were significantly reduced. Thus, while for chitosan about 840microg/ml reducing sugar was produced in 4h time, and 780microg/ml was produced for diisocyanatohexane crosslinked chitosan, only 400microg/ml and 320microg/ml reducing sugars were produced for cadmium sulfate with crosslinked chitosan and diisocyanatohexane crosslinked chitosan, respectively. Similar results are obtained for other crosslinking agents. Studies on preincubation of the metal with the enzyme show that of the metals studied, Mn has no effect on preincubatioin with the enzyme, Hg, Cd, Pb, and Cu completely deactivates the enzyme, while Zn reduces the enzyme activity by about 43.3%. Preincubation of the metal salts with the chitosan shows that Hg and Cu completely deactivate the molecule from enzyme hydrolysis, Cd and Zn inactivate it to the extent of 56.8% and 43.3%, respectively, while Mn has no effect. Availability of the amino functions seems to be a key feature for the chitosanase to hydrolyze the chitosan polymer. This was also proved by the significant increase in the extent of hydrolysis for chitosan samples with 88% (final value 1120micro/ml reducing sugar) and 85% deacetylation (final value 840microg/ml reducing sugar). HPIC studies of the products show that a variety of oligomers are produced in the chitosanase enzyme hydrolytic reaction.

Strain improvement of Penicillium janthinellum NCIM 1171 for increased cellulase production.

The strain of Penicillium janthinellum NCIM 1171 was subjected to mutation involving treatment of Ethyl Methyl Sulfonate (EMS) for 24h followed by UV-irradiation for 3min. Successive mutants showed enhanced cellulase production (EMS-UV-8), clearance zone on Avicel containing plate (SM2) and rapid growth on Walseth cellulose agar plates containing 0.2% 2-deoxy-d-glucose (SM3). These mutants were transferred to Walseth cellulose plates containing higher concentration (1.5%) of 2-deoxy-d-glucose (SM4) in which only five mutants showed clearance zone on SM4. All these mutants showed approximately two-fold increase in activity of both FPase and CMCase in shake flask culture when grown on basal medium containing CP-123 (1%) and wheat bran (2.5%). The enzyme preparations from these mutants were used to hydrolyze Avicel. Higher hydrolysis yields of Avicel were obtained with enzyme preparations of EU1. This is the first report on the isolation and selection of mutants based on hydrolysis of Avicel, which is the most crystalline substrate.

Production of lactic acid and fructose from media with cane sugar using mutant of Lactobacillus delbrueckii NCIM 2365.

AIMS: To examine the potential of Lactobacillus delbrueckii mutant, Uc-3 to produce lactic acid and fructose from sucrose-based media. METHODS AND RESULTS: The mutant of L. delbrueckii NCIM 2365 was cultivated in shake flask containing hydrolysed cane sugar (sucrose)-based medium. The lactic acid yield and volumetric productivity with hydrolysed cane concentration up to 200 g l(-1) were in the range of 92-97% of the theoretical value and between 2.7 and 3.8 g l(-1) h(-1), respectively. The fructose fraction of the syrup produced was more than 95% when the total initial sugar concentration in the medium was higher (150-200 g l(-1)). There are no unwanted byproducts detected in the fermentation broth. CONCLUSIONS: We demonstrated that L. delbrueckii mutant Uc-3 was able to utilize glucose preferentially to produce lactic acid and fructose from hydrolysed cane sugar in batch fermentation process. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings will be useful in the production of lactic acid and high fructose syrups using media with high concentrations of sucrose-based raw materials. This approach can lead to modification of the traditional fermentation processes to obtain value-added byproducts, attaining better process economics.

Chemoenzymatic synthesis of D(-)phenylglycine using hydantoinase of Pseudomonas desmolyticum resting cells.

We screened 125 Pseudomonas strains from our culture collection for the production of hydantoinase activity using DL-phenylhydantoin as a substrate. Pseudomonas desmolyticum NCIM 2112 was found to be the best hydantoinase (dihydropyrimidinase E.C. 3.5.2.2) producer. The enzymatic reactions were carried out using 18-20-h grown cells in nutrient broth and 5-phenylhydantoin as the substrate. Optimization studies for the biotransformation reaction were performed to increase product yield. The optimum pH and temperature for D(-)N-carbamoylphenylglycine production were 9.5 and 30 degrees C, respectively. Biotransformation under these alkaline conditions allowed the complete conversion of 27.0 g l-1 of DL-phenylhydantoin to 26.5 g l-1 of N-carbamoylphenylglycine within 24 h, with a molar yield of 90%. The hydantoinase involved in this biotransformation process was strictly D-stereospecific, because the product isolated was pure D(-)N-carbamoylphenylglycine. This pure product was further chemically converted to D(-)phenylglycine using nitrous acid with an 80% chemical yield. Thus, the overall conversion efficiency of DL-5-phenylhydantoin to D(-)phenylglycine was found to be 65-68%.

Industrial yeast strain improvement: construction of a highly flocculent yeast with a killer character by protoplast fusion.

Conditions were optimized for rapid release and improved regeneration of protoplasts of Saccharomyces cerevisiae NCIM 3458. Rapid protoplast release was also obtained with representatives of several other yeast genera under the modified conditions of treatment. The application of the procedure in construction of a highly flocculent Saccharomyces cerevisiae with a killer character is described. Fusion was effected between UV-killed protoplasts of S. cerevisiae NCIM 3578 with a killer character and live protoplasts of the highly flocculent S. cerevisiae NCIM 3528 in the presence of polyethylene glycol (PEG) 6000. Fusants were selected using benomyl resistance as marker, the killer toxin producer rather than the highly flocculent yeast being resistant to the fungicide at a concentration of 100 micrograms ml-1. Fusants were also characterized by their DNA contents, capacity for ethanolic fermentation of molasses sugar and levels of invertase, alcohol dehydrogenase and pyruvate decarboxylase activities.

Protoplast fusion: a tool for intergeneric gene transfer in bacteria.

Protoplasts can be isolated from bacterial cells by digestion of the cell wall with the help of lysozyme in presence of osmotic stabilizers. Fusion of protoplasts can be induced by chemical fusogens like polyethylene glycol. The electrofusion technique has been reported in bacteria in which the fusion frequency is much higher than that obtained by PEG induced protoplast fusion. This technology allows recombination to take place not only between related species but also between unrelated genera and is of great potential in the breeding and improvement of industrial strains. This review includes the information and developments on the protoplast fusion in bacteria with special reference to genetic recombination by protoplast fusion between phylogenetically unrelated bacteria.

Protection of Aspergillus niger cellulases by urea during growth on glucose or glycerol supplemented media.

The cellulase enzymes of Aspergillus niger were found to undergo catabolite repression in the presence of glucose and glycerol accompanied by sudden drop in pH of the fermentation medium below 2.0. This sudden drop in pH caused inactivation of cellulolytic enzymes produced by Aspergillus niger. The supplementation of nitrogen sources, especially urea, protects A. niger cellulases from inactivation caused by a sudden drop in pH, since urea helped to maintain the pH of the fermentation medium between 3.5 and 4.5. The role of urea in the protection of cellulase was more prominent when it was used in combination with glycerol (5%).

Optimization of cellulase production by Aspergillus niger NCIM 1207.

Aspergillus niger NCIM 1207 produces high levels of extracellular beta-glucosidase and xylanase activities in submerged fermentation. Among the nitrogen sources, ammonium sulfate, ammonium dihydrogen orthophosphate, and corn-steep liquor were the best for the production of cellulolytic enzymes by A. niger. The optimum pH and temperature for cellulase production were 3.0-5.5 and 28 degrees C, respectively. The cellulase complex of this strain was found to undergo catabolite repression in the presence of high concentrations of glucose. Glycerol at all concentrations caused catabolite repression of cellulase production. The addition of glucose (up to 1% concentration) enhanced the production of cellulolytic enzymes, but a higher concentration of glucose effected the pronounced repression of enzymes. Generally the growth on glucose- or glycerol-containing medium was accompanied by a sudden drop in the pH of the fermentation medium to 2.0.

Association of logistic and Poisson models of infection with some physical characteristics of a single component plant virus.

A logistic model was recently formulated to describe the relationship between concentration of a single component plant virus and infections produced by inoculation to a local lesion host. In this paper the logistic is combined with a Poisson model. The logistic makes accurate fitting possible for a variety of infection-dilution series; and the Poisson acts as a base line, indicating whether lesion numbers are compatible with the hypothesis that random infection of similar infection sites has occurred. A logarithmic form of the logistic equation gives a straight line with negative slope (logit slope) which is useful in characterizing dilution series to which the logistic is fitted. A modified Poisson equation can also be fitted to a range of dilution series; it provides an independent estimate of slope for curves not widely divergent from the standard Poisson. Models have also been developed to define the limits of concentration within which single virions are likely to be randomly dispersed in inoculum without immediate contact with other virions, and are therefore more likely to enter inoculated tissue independently and cause random infections. Models are formulated for aggregation of tobacco mosaic virus in monolayers, crystals, and lenticular aggregates. Published and unpublished data are fitted and analyzed using some of these models.

Differential Expression of Xylanases and Endoglucanases in the Hybrid Derived from Intergeneric Protoplast Fusion between a Cellulomonas sp. and Bacillus subtilis.

A stable hybrid obtained by protoplast fusion between a Cellulomonas sp. and Bacillus subtilis exhibits an altered pattern of enzyme induction with different cellulosic substrates. Unlike in the Cellulomonas sp., xylanase was induced in the hybrid organism specifically by xylan, and endoglucanase was induced by carboxymethyl cellulose. The amount and specific activity of xylanase produced by the hybrid were more than those produced by the Cellulomonas sp. beta-Glucosidase which is cell bound or intracellular in the Cellulomonas sp. was secreted by the hybrid organism, and relative amounts of extracellular beta-glucosidase were high. Furthermore, this extracellular beta-glucosidase activity was dependent on the nature of the cellulosic substrate. Endoglucanases synthesized in the hybrid differed in their electrophoretic mobilities as compared with the parental enzymes.

Confirmation and certainty in toxicology screening.

Confirmation of presumptive positive urine drug screens, necessary to minimize the reporting of false-positive results, can be costly and time-consuming. The predictive value model can be used to select the confirming tests and to calculate the confidence of the result. The predictive value of a test result is the probability, based on the sensitivity and specificity of the test, that the result is a true positive or a true negative. The predictive value model applied to toxicology screening tests for drugs of abuse showed that prevalence, in addition to sensitivity and specificity, was the factor controlling the confidence level of a result. For example, the predictive value of a positive result for a screening test that has a sensitivity of 99% and a specificity of 99%, applied to screening in a population with a prevalence of 1% is 0.50; for a prevalence of 10%, it is 0.92. Confirmation with a second, chemically independent, test of equal sensitivity and specificity increases the predictive value to 0.99.

Relationship between plant virus concentration and infectivity: a 'growth curve' model.

A logistic ('growth curve') model is formulated and applied to the relationship between numbers of infections (local lesions) produced by a virus on inoculated plants and the concentration of the virus in the inoculum. This model has the advantages of being simple, data-based and therefore not founded on limiting postulates, and of being applicable to a wide range of infection-dilution series, including those obtained from multicomponent viruses. Here, it is applied to common tobacco mosaic virus. Examples of infection-dilution series taken from the literature are fitted more closely and more objectively than they were fitted by the original authors. A limiting number for lesions is estimated by minimizing chi 2 values for differences between observed lesion numbers and those calculated from the logistic equation. The complete fitting procedure can be programmed on a hand-held calculator.