Papers by K. MADHAVAN NAMPOOTHIRI
Journal of Biological Chemistry, 2001
Malonyl coenzyme A (CoA)-acyl carrier protein (ACP) transacylase (MCAT) is an essential enzyme in... more Malonyl coenzyme A (CoA)-acyl carrier protein (ACP) transacylase (MCAT) is an essential enzyme in the biosynthesis of fatty acids in all bacteria, including Mycobacterium tuberculosis. MCAT catalyzes the transacylation of malonate from malonyl-CoA to activated holo-ACP, to generate malonyl-ACP, which is an elongation substrate in fatty acid biosynthesis. To clarify the roles of the mycobacterial acyl carrier protein (AcpM) and MCAT in fatty acid and mycolic acid biosynthesis, we have cloned, expressed, and purified acpM and mtfabD (malonyl-CoA: AcpM transacylase) from M. tuberculosis. According to the culture conditions used, AcpM was produced in Escherichia coli in two or three different forms: apo-AcpM, holo-AcpM, and palmitoylated-AcpM, as revealed by electrospray mass spectrometry. The mtfabD gene encoding a putative MCAT was used to complement a thermosensitive E. coli fabD mutant. Expression and purification of mtFabD resulted in an active enzyme displaying strong MCAT activity in vitro. Enzymatic studies using different ACP substrates established that holo-AcpM constitutes the preferred substrate for mtFabD. In order to provide further insight into the structure-function relationship of mtFabD, different mutant proteins were generated. All mutations (Q9A, R116A, H194A, Q243A, S91T, and S91A) completely abrogated MCAT activity in vitro, thus underlining the importance of these residues in transacylation. The generation and characterization of the AcpM forms and mtFabD opens the way for further studies relating to fatty acid and mycolic acid biosynthesis to be explored in M. tuberculosis. Since a specific type of FabD is found in mycobacterial species, it represents an attractive new drug target waiting to be exploited.
Biochemical Journal, 2002
Mycolic acids consist of long-chain α-alkyl-β-hydroxy fatty acids that are produced by successive... more Mycolic acids consist of long-chain α-alkyl-β-hydroxy fatty acids that are produced by successive rounds of elongation catalysed by a type II fatty acid synthase (FAS-II). A key feature in the elongation process is the condensation of a two-carbon unit from malonyl-acyl-carrier protein (ACP) to a growing acyl-ACP chain catalysed by a β-ketoacyl-ACP synthase (Kas). In the present study, we provide evidence that kasA from Mycobacterium tuberculosis encodes an enzyme that elongates in vivo the meromycolate chain, in both Mycobacterium smegmatis and Mycobacterium chelonae. We demonstrate that KasA belongs to the FAS-II system, which utilizes primarily palmitoyl-ACP rather than short-chain acyl-ACP primers. Furthermore, in an in vitro condensing assay using purified recombinant KasA, palmitoyl-AcpM and malonyl-AcpM, KasA was found to express Kas activity. Also, mutated KasA proteins, with mutation of Cys171, His311, Lys340 and His345 to Ala abrogated the condensation activity of KasA in ...
Frontiers in Bioengineering and Biotechnology, 2021
This research topic aimed to introduce the current advancements in the biosynthesis of amino acid... more This research topic aimed to introduce the current advancements in the biosynthesis of amino acids and their derived products from renewable feedstocks. Authors were invited to contribute original research and review articles that provided a comprehensive discussion and analysis of the current success and future outlooks for biosynthesis of various amino acids and derived chemicals. A total of 12 manuscripts were submitted, and 11 were accepted for publication after a thorough and rigorous peer review process. The papers were selected in such a way to give a flavor of a variety of topics related to the production of amino acids and their derived products, including biological and chemical catalytic production, design and construction of new molecular pathways for amino acid production, application of metabolic engineering and synthetic biology strategies, as well as the utilization of non-food renewable feedstock such as rice straw. We believed that the papers in this research topic would bring readers the latest advances in these fields. Bioproduction of 5-aminovalerate (5AVA) from renewable feedstock could support a sustainable biorefinery process to produce bioplastics. The paper by Cheng et al. developed a promising artificial pathway for the efficient 5AVA synthesis by establishing a 2-keto-6aminocaproate-mediated pathway. Introduction of L-lysine α-oxidase from Scomber japonicas, α-ketoacid decarboxylase from Lactococcus lactis and aldehyde dehydrogenase from Escherichia coli could finally achieved the biosynthesis of 5AVA from L-lysine with the high titre through the fed-batch fermentation. Another paper by Cheng et al. further presented an efficient biobased co-production of 5AVA and δ-valerolactam in E. coli from L-lysine. With the optimized cultivation conditions, the titers of 5AVA and δ-valerolactam were improved, and their ratio was identified to be affected by pH values. The paper by Sasikumar et al. established the production of 5AVA and putrescine from the biomass-derived sugars by using the engineered Corynebacterium glutamicum strain. It was indicated that with the heterologous introduction of genes xylA Xc and xylB Cg , the modified strain could co-produce putrescine and 5-AVA by consuming a blend of glucose and xylose. Further investigation by using alkali-hydrolases pretreated rice straw hydrolysate (RSH) as the raw material also yielded the generation of putrescine and 5AVA. The paper by Brito et al. demonstrated another study involved in the methanol-based production of 5AVA using genetically modified Bacillus methanolicus. Five different metabolic pathways were evaluated, whereof two directly converted L-lysine to 5AVA and three used cadaverine as an intermediate. The results indicated the proof-of-concept 5AVA production from methanol at 50 °C, enabled by two pathways out of the five tested with the
Biochemical Engineering Journal, Oct 1, 2007
Optimization of five process parameters (concentrations of cassava bagasse, enzyme, yeast extract... more Optimization of five process parameters (concentrations of cassava bagasse, enzyme, yeast extract, NH 4 Cl and inoculum) was attempted using a Box-Behnken design for the optimal production of l(+)-lactic acid by a mixed culture of Lactobacillus casei and Lactobacillus delbrueckii by simultaneous saccharification and fermentation. Maximum lactic acid yield of 81 g/L was obtained when 15% (w/v) cassava bagasse treated with 12.5 mL/L enzyme mixture was supplemented with 7.5 g/L yeast extract and 3 g/L NH 4 Cl and inoculated with 3 × 10 10 CFU/L of lactobacilli and incubated for 60 h at 37 • C as static culture.
CRC Press eBooks, Jul 21, 2010
Applied Biochemistry and Biotechnology, Oct 13, 2010
Aminopeptidases catalyze the cleavage of specific amino acids from the amino terminus of protein ... more Aminopeptidases catalyze the cleavage of specific amino acids from the amino terminus of protein or peptide substrates. A proline-specific aminopeptidase was purified to homogeneity from the culture-free extract of Streptomyces lavendulae ATCC 14162 in sequential steps comprising ammonium sulfate precipitation, ultra-filtration, and column chromatography on Q-sepharose and Sephadex G-100. The purified protein showed approximately 60 kDa in SDS-PAGE and was optimally active at pH 6.5 and 40°C. Kinetic studies showed a K m and V max of 0.23 mM and 0.087 μmol/min, respectively, using Prop -NA, the substrate with maximum specificity. Enzyme activity was inhibited by PMSF and ions like Zn 2+ , Co 2+ , and Ni 2+. However, unlike other aminopeptidases, the activity was enhanced in the presence of DTT, 1,10-phenanthroline, EDTA, amastatin, and bestatin. Ions like Ca 2+ , Mg 2+ , and Mn 2+ also enhanced the activity.
Journal of Basic Microbiology, Feb 1, 2007
A process based on low cost production media was developed for fermentative production of L(+) la... more A process based on low cost production media was developed for fermentative production of L(+) lactic acid. Various process parameters were optimized for the production of L(+) lactic acid from cassava starch hydrolyzate-based medium by immobilized whole cells of Lactobacillus delbrueckii. The parameters considered for optimization were concentration of alginate and CaCl 2 , initial cell concentration, curing time, bead size and incubation time of fermentation. Reusability of immobilized cells up to six batches was observed without any decline in lactate production. Lactate yield for L. delbrueckii was 0.93 g lactic acid × g-1 reducing sugar with a production rate of 0.33 g × l-1 × h-1. A column bioreactor packed with immobilized L. delbrueckii was run for three weeks in a continuous mode with a lactate yield of 0.75-0.95 g lactic acid × g-1 reducing sugar with an average production rate of 0.48 g × l-1 × h-1. The study revealed that agro residues like cassava bagasse could be used as source material for developing a low cost technology for lactate production.
Journal of Applied Microbiology, Jul 1, 2008
Aim: Molecular cloning, overexpression and biochemical characterization of the genes from the Myc... more Aim: Molecular cloning, overexpression and biochemical characterization of the genes from the Mycobacterium tuberculosis H37Rv genome having hypothetical b-lactamases activity. Methods and Results: Analysis of the M. tuberculosis H37Rv genome revealed that Rv2068c, Rv0406c and Rv3677c gene products were predicted to exhibit b-lactamases activity. All the three genes were cloned in pET28a vector and overexpressed in C41 (DE3) Escherichia coli cells. The His-tagged recombinant proteins were confirmed by immunoblotting and were shown to have b-lactamase activity by the hydrolysis of nitrocefin and other b-lactams. Catalytic parameters for all the recombinant proteins were derived followed by the enzyme inhibition studies. Antibiotic susceptibility studies using the recombinant strains showed an increased resistance against different classes of b-lactam antibiotics. Conclusion: The study revealed the possibility of more than one gene in M. tuberculosis, encoding proteins having b-lactamase or b-lactamase-like activity, giving wide spectrum of resistance against b-lactams. Significance and Impact of the Study: Systematic study of hypothetical b-lactamases of M. tuberculosis and related species and their correlation with b-lactam and inhibitor susceptibility profile might be useful in developing new antibiotic regime for the treatment of tuberculosis caused by multiple drug resistant (MDR) strains.
Biochemical Engineering Journal, Oct 1, 2015
Enzyme technology is a well established branch of the biochemical science which is going through ... more Enzyme technology is a well established branch of the biochemical science which is going through a phase of maturation as well as evolution. The maturation is shown by the development of the theory, their function and through the formation and configuration of their three-dimension structure, in this era of global industrialization. A better understanding enzymes and their functional significance suggests many novel applications for these catalytic activities and for continual discovery with novel properties by R&D. They are being used on an industrial and research scale for the variety of reactions catalyzed with environmental conditions. This is just the start of the industrial enzyme era which is preparing itself to put known enzymes to novel uses and novel enzymes, discovered or tailored, to catalyze unexploited reactions. As the demand is for cleaner and greener technology to preserve our mother earth for our descendant, the use of enzymes that can replace harmful chemical reactions are extremely importance and most of the current R&D on enzymes is directed towards this issue. Similarly use of enzymes in extreme harsh conditions such as high and low temperatures and pH are also more prevalent. Hence the evaluation of present R&D directions on industrial enzymes seems to be important and this paper assesses the status of industrial enzyme research globally as well as in Indian context with use in various industries, it application, the present status of R&D and commercialization. Since enzymes are now widely used in biotransformation and considering the importance of enzymes in the synthesis of chiral molecules of pharmaceutical importance, a separate section on enzymes involved in biotransformation are also reviewed in detail. This study is based on the search from scientific databases such as SciVerse Scopus, Google, other web sources, etc.
Bioresource Technology, 1998
Studies were performed to elucidate the optimal conditions (gel concentration, bead size, initial... more Studies were performed to elucidate the optimal conditions (gel concentration, bead size, initial biomass, storage period) for immobilization of growing cells of Brevibacterium sp. (DSM 20411) in alginate and agar gels and to evaluate the immobilized biocatalyst in batch ...
Elsevier eBooks, 2017
Abstract The concept of biodegradable plastics is receiving significant attention with respect to... more Abstract The concept of biodegradable plastics is receiving significant attention with respect to solid waste accumulation. Enormous efforts have been made in revolutionizing degradable biological materials without any environmental pollution to substitute for oil-based traditional plastics. With the overuse of plastics and increasing pressure being placed on capacities available for plastic waste disposal, the requirement for biodegradable plastics and biodegradation of plastic wastes has obtained immense importance in recent years. Awareness of the impact on the environmental waste problem has awakened research in the area of degradable polymers. Considering the emerging environmental issues, the demand to develop a material that does not burden nature has increased. It is essential to study the microbial degradation of biopolymers to understand what is necessary for biodegradation and the mechanisms involved. Widespread investigations on the biodegradation of plastics have been carried out to overcome the environmental problems associated with synthetic plastic waste. This chapter provides an overview of the degradation mechanisms of biodegradable polymers with particular emphasis on the main parameters affecting the degradation of polymeric biomaterials.
Applied Biochemistry and Biotechnology, 2006
Saccharification and fermentation of cassava (Manihot esculenta) bagasse was carried out in a sin... more Saccharification and fermentation of cassava (Manihot esculenta) bagasse was carried out in a single step for the production of L-(+)-lactic acid by Lactobacillus casei and Lactobacillus delbrueckii. Using 15.5% w/v of cassava bagasse as the raw material, a maximum starch to lactic acid conversion of 96% was obtained with L. casei with a productivity rate of 1.40 mg/mL•h and maximum yield of 83.8 mg/mL. It was 94% with L. delbrueckii with a productivity rate of 1.36 mg/mL•h and maximum yield of 81.9 mg/mL. Supplementation of bagasse with 0.01% w/v MnCl 2 showed positive influence on the lactic acid production by L. casei.
Journal of Basic Microbiology, 1995
(Biotechnology Unit, Regional Research Laboratory, Trivandrum - 695019, India) ... Effect of diff... more (Biotechnology Unit, Regional Research Laboratory, Trivandrum - 695019, India) ... Effect of different carbon sources on growth ... Experiments were carried out to find the efficiency of Brevibacterium sp. to utilize different carbon sources (glucose, fructose, sucrose, maltose, lactose, xylose ...
Process Biochemistry, Jun 1, 2003
The biopolymer gellan belongs to the family of microbial polysaccharides having a wide range of i... more The biopolymer gellan belongs to the family of microbial polysaccharides having a wide range of industrial applications. Attempts were made to produce gellan gum in submerged fermentation (SmF) using Sphingomonas paucimobilis ATCC 31461. Studies on the nutritional ...
Humana Press eBooks, 2005
Summary Proteases represent one of the three largest groups of industrial enzymes and account for... more Summary Proteases represent one of the three largest groups of industrial enzymes and account for about 60% of the total worldwide sale of enzymes. They are degradative enzymes of central importance because they can be employed in a number of industries to create change in ...
Food Technology and Biotechnology, Aug 10, 2011
The productivity of enzyme fermentations depends critically on maintaining a high oxygen transfer... more The productivity of enzyme fermentations depends critically on maintaining a high oxygen transfer rate to satisfy the optimal oxygen demand of the microorganism for product formation. Among the several factors that affect oxygen transfer rates in a fermentor are the air flow rate and agitation. The production of a-amylase by Bacillus amyloliquefaciens was performed in 600-mL and 5-litre fermentor with a working volume of 300 mL and 3 L, respectively. The experiments indicated a requirement of high rates of aeration to enhance the enzyme yield. The biomass yield and productivity of the enzyme were found to have a linear relationship with the air flow rate, and the highest productivity was observed at 1.0 vvm. A maximum productivity of 41.4 U/(mL•h) was obtained after 14 h of fermentation in 600-mL fermentor system and a comparable productivity of 40 U/(mL•h) was obtained after 12 h in the 5-litre fermentor.
Frontiers in Microbiology, 2022
Graphical Strategy to valorize non-utilized nitrogen from sidestreams by producing nitrogen-conta... more Graphical Strategy to valorize non-utilized nitrogen from sidestreams by producing nitrogen-containing products.
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Papers by K. MADHAVAN NAMPOOTHIRI