I. M. Santha

In vitro kinetics of soybean lipoxygenase with combinatorial fatty substrates and its functional significance in off flavour development

Lipoxygenase (Lox) mediated oxidation of polyunsaturated fatty acids (PUFA) in mature soya seeds results in objectionable flavour. In the present study, Lox isozymes were purified to near homogeneity (107-fold). Lox-2 and 3 displayed remarkable kinetic preference (1.7 and 1.5-fold, respectively) for low PUFA ratios (LA/LeA) (PRs) among the selected PUFA combinations. Lox-1 displayed no specific preference. Pure Lox-1 displayed unbiased response towards substrates with marginal preference (1.2-fold) for linoleic acid at its optimum pH. Volatile compounds profiling showed a direct relationship between PRs and hexanal to trans-2-hexenal (1.47, 2.24 and 18.90 for 2, 7 and 15 PRs, respectively) ratio. The off-flavour determining parameters like TBA value, carbonyl value and lipid hydroperoxides (LHPODs) exhibited significant negative correlation (0.76, 0.74, 0.72; p<0.0001) in selected soya genotypes displaying varied PRs and significant positive correlation (0.89, 0.81. 0.89; p<0.0001) with ratio of PI (polyene index) to PRs - suggesting the plausible significance of PUFA ratios in biological lipid peroxidation.

Metabolism of the Lathyrus sativus L. Neurotoxin, β-N-Oxalyl-L-α, β-diaminopropionic Acid, by a Pure Culture of a Soil-borne Microbe

Pure cultures of bacteria capable of utilizing the Lathyrus sativus L. neurotoxin, β-N-oxalyl-L-α, β-diaminopropionic acid (ODAP), as their sole carbon and nitrogen source have been isolated from soil-sludge filtrates. Three independent isolates, designated BYA1, BYT1, and BYK1, were selected by repetitive growth on the neurotoxin and purified based upon their antibiotic resistance. Of the three Isolates, strain BYA1 demonstrated the highest capacity for ODAP utilization, degrading greater than 98% of the ODAP present In the culture media within 12 h. Using a variety of morphological and biochemical criteria BYA1 was Identified as an Enterobacter cloacae. The bacterium harbors a single large plasmid (designated pBYA1) approximately 40–50 kb in size that contains the genetic Information for ODAP utilization and antibiotic resistance. Transformation experiments with E. coli recipient strains were used to further define the location of the sequences involved in ODAP metabolism.

Cloning and Characterization of a Gene Responsible for the Lathyrus sativus Neurotoxin Degradation

A plasmid pBYA1, capable of degrading the Lathyrus sativus neurotoxin β-N-oxalyl amino alanine (BOAA), was isolated from a soli microbe and a physical map of the plasmid constructed. A partial Sau3A library of the plasmid was then prepared in pUC18 and a recombinant clone with a 1.8 kb insert capable of growing in M9 medium, with BOAA as sole source of carbon and nitrogen, was isolated. A nested set of deletions of this 1.8 kb fragment was then generated using Bal31 and the shortened fragments subcloned in the vector pUC18. Each of these clones were sequenced by Sanger’s dideoxy method and the sequences overlapped and analysed. The 1.8 kb BOAA degrading fragment was found to contain a 630 nucleotide long coding stretch, coding for 191 amino acids. The initial 56 nucleotides were found to contain sequences resembling the Pribnow-Schaller box of prokaryotes, the “-35” sequences and a sequence resembling the “-43” region of the lac promoter.

RAPD Analysis of Lathyrus sativus Somaclones

RAPD patterns were studied in seven somaclones of Lathyrus sativus having contrasting characteristics alongwith the parent cultivar P-24. Out of 81 decamer random primers used, 5 did not amplify and 24 revealed DNA polymorphism while the rest generated monomorphic banding patterns. Eight unique bands were amplified with different primers in four different somaclones. With most of the informative primers differences were observed between somaclones and also between some of the somaclones and parent cultivar P-24. More than 90% similarity in the RAPD patterns was evident among the somaclones and the parent cultivar P-24. Though it was not possible to identify a particular somaclone with a single primer, a combination of two or more primers could be employed to identify a somaclone.

Molecular Response to Water Stress in Lathyrus sativus

Studies on response of Lathyrus sativus plants to water stress showed that the plants recovered to pre-stressed level if revived within first seven days of stress. After 12 days of stress period, root weight of stressed plants was 10 times lesser than control. Lipoxygenase expression was the highest in leaves followed by very low levels in stems and undetectable in roots. Lox A and B messengers were respectively 3 and 1.5 fold more in stressed leaves than control. A −22kD polypeptide was observed in stressed plants on SDS-PAGE.

Physiological and Biochemical Analysis of Somaclones Derived from Leaf Explants of Lathyrus sativus

Low ODAP somaclones have been evaluated for physiological and biochemical parameters especially in relation to attributes that lead to increased biomass production. All the somaclones during development had substantially lower ODAP content in leaves as compared to parent P24. Considerable variation was observed in relation to leaf width, leaf length, internodal length and leaf area. Somaclone Bio L12 had the highest whereas parent P24 and Bi0164 had the least leaf area. Harvest index was the highest and biomass production was the lowest in the Bio 164. Bio L08 gave the highest seed yield. Photosynthetic rates were also higher in Bio L12, although no significant positive correlation was observed in leaf photosynthesis and seed yield. The differences in physiolpgical and biochemical parameters indicate the possibility of development of high yielding genotypes. The results in present investigation show differences in photosynthetic rate, leaf characteristics, seed yield and ODAP content among somalones and parent. Somaclones with extremely low ODAP content with variability in leaf morphology and photosynthetic rate is indicative of variation induced during plant tissue culture.

Cloning of ODAP Degrading Gene and Its Expression as Fusion Protein in Escherichia coli

A gene responsible for the degradation of ß-N-Oxalyl diaminopropionic acid (ODAP) was fused to the maIE gene, which codes for maltose binding protein, by cloning into an expression vector pMAL c2. The gene has been expressed as fusion protein of mol wt approximately 62 kD. It has been purified by affinity chromatography. The fusion protein has been cleaved by an endoprotease factor Xa and the presence of maltose binding protein and the product of the cloned gene confirmed. SDS-PAGE has shown that the product of the ODAP degrading gene is a single polypeptide of mol wt of about 20.7 kD.

Cloning of a Gene Sequence for co-3 Desaturase from Brassica juncea

AbstractcDNA was synthesized from RNA isolated from developing seeds of Brassica juncea cv Pusa Jai Kisan at 25–45 days after flowering and partial cDNA sequences encoding ω-3 desaturase gene were PCR amplified using specifically designed primers. The amplified products were cloned, and three of the clones subjected to partial sequence analysis. The clones showed high homology to ω-3 desaturase sequence from other sources. A genomic clone corresponding to ω-3 desaturase was isolated from the genomic library of B. juncea constructed in λEMBL-3 vector using one of the PCR clones.

Molecular Characterisation of Low ODAP Somaclones of Lathyrus sativus

Somaclones of Lathyrus sativus having low ODAP contents were characterised at molecular level with respect to soluble protein pattern, esterase and ADH isozyme pattern, Southern hybridisation of genomic and mitochondrial DNA with specific probes. In 10-day-old seedling esterase isozyme pattern showed the presence of an unique band of Rm 0.73 in Bio L-12 and 15-8-1 and the absence of a band of Rm 0.80 in 15-8-1, as compared to parent and other somaclones. Southern hybridisation of genomic DNA with cDNA clone 29 showed an extra band of 13.5 kb in L-56 and 15-8-1 only. Hybridisation of mitochondrial DNA with mitochondrial specific ati ATPase probe showed differences with the pattern from Bio R-15 being unique. Some of these differences observed will be useful as marker for their identification.

Molecular Cloning and Characterization of Oleoyl-ACP Thioesterase Gene from Brassica juncea

Oleoyl-ACP thioesterase (TE) is the chain length-determining enzyme in de novo biosynthesis of oleic acid. For cloning the gene encoding oleoyl-ACP TE from Brassica juncea, a PCR-amplified DNA probe was developed with the primers designed from the known sequences available in the Genbank. It was used to screen a genomic library of B. juncea constructed in λEMBL 3 to get the complete gene. A 4.0 kb Bam HI fragment from the clone X5.12 and hybridizing to the probe was sequenced. A stretch of 2606 by genomic sequence was found to contain an ORF of 1101 by with ∼ 0.4 kb untranslated regions at both the ends and 5 introns of varied lengths. By homology analysis, the ORF was found to encode an oleoyl-ACP thioesterase gene. The promoter region contains major cis elements required for transcription.