Farooq Latif

Isolation, partial characterization, and the effect of plant growth-promoting bacteria (PGPB) on micro-propagated sugarcane in vitro

We report the isolation of nitrogen fixing, phytohormone producing bacteria from sugarcane and their beneficial effects on the growth of micropropagated sugarcane plantlets. Detection of the nitrogen fixing bacteria by ARA-based MPN (acetylene reduction assay-based most probable number) method indicated the presence of up to 106 bacteria per gram dry weight of stem and 107 bacteria per gram dry weight of root of field-grown sugarcane. Two nitrogen fixing bacterial isolates were obtained from stem (SC11, SC20) and two from the roots (SR12, SR13) of field-grown plants. These isolates were identified as Enterobacter sp. strains on the basis of their morphological characteristics and biochemical tests. The isolate SC20 was further characterized by 16S rRNA sequence analysis, which showed high sequence similarity to the sequence of Enterobacter cloacae and Klebsiella oxytoca. All the isolates produced the phytohormone indoleacetic acid (IAA) in pure culture and this IAA production was enhanced in growth medium containing tryptophan. The bacterial isolates were used to inoculate micro-propagated sugarcane in vitro where maximum increase in the root and shoot weight over control was observed in the plantlets inoculated with strain SC20. By using the15N isotope dilution technique, maximum nitrogen fixation contribution (28% of total plant nitrogen) was detected in plantlets inoculated with isolate SC20.

Innovative kinetic and thermodynamic analysis of a purified superactive xylanase from Scopulariopsis sp.

Two isoenzymes of endo-1,4-β-xylanase (EC 3.2.1.8) from Scopulariopsis sp. were purified by a combination of ammonium sulfate precipitation, hydrophobic interaction, and anion-exchange and gel filtration chromatography. The native mol wts of the least acidic xylanase (LAX) and the highly acidic xylanase (HAX) were 25 and 144 kDa and the subunit mol wts were 25 and 36 kDa, respectively. The kcat values of LAX and HAX for oat-spelt xylan at 40°C, pH 6.5, were 95,000 and 9900 min−1 and the Km values of LAX and HAX were 30 and 3.3 mg/mL. The thermodynamic activation parameters of xylan hydrolysis showed that the high activity of LAX when compared with HAX was not owing to a reduction in ΔH# but was entropically driven. High-performance liquid chromatography analysis of the degradation products showed that LAX formed both xylotrioses and xylobioses, but HAX predominantly formed xylotrioses. The half-lives of LAX and HAX at 50°C in 50 mM 2-N-morpholino ethanesulfonic acid (MES), pH 6.5 buffer were 267 and 69 min, respectively. Thermodynamic analysis showed that at lower temperatures, the increased thermostability of LAX (ΔH#=306 kJ/mol) compared with HAX (ΔH#=264 kJ/mol) was owing to more noncovalent surface interactions. At higher temperatures, LAX (ΔS*=−232 J/[mol·K]) was more thermostable than HAX (ΔS*=490 J/[mol·K]) owing to a more ordered transition-state conformation. An energy-activity diagram was introduced showing that kcat/Km does not successfully explain the true kinetic behavior of both xylanase isoenzymes. The simultaneously thermostable and highly active LAX could be utilized in biotechnological processes involving xylan hydrolysis.

Identification of plant growth hormones produced by bacterial isolates from rice, wheat and kallar grass

Identification and quantification of the plant growth hormones indoleacetic acid and gibberellic acid, produced by plant growth-promoting rhizobacteria (PGPR), was carried out by using high-pressure liquid chromatography (HPLC). The PGPR strains were isolated from roots of rice, wheat and kallar grass and belonged to the genera Azoarcus, Azospirillum, Enterobacter, Pseudomonas and Zoogloea. For these studies, bacteria were grown in liquid nitrogen free malate (NFM) or combined carbon medium (CCM) containing tryptophan and combined nitrogen. Some Azospirillum strains produced both indoleacetic acid and gibberellic acid, while none of the Enterobacter spp. tested produced these growth hormones. Azoarcus strain K-1 produced higher amounts of gibberellic acid and Azospirillum strain ER-2 produced higher amounts of indoleacetic acid. Indoleacetic acid production increased with the age of bacterial cultures while a decrease in the production of gibberellic acid was noted at later growth stages. Pure indoleacetic acid and gibberellic acid in the concentration range 1–2 µg/ml increased root area and plant biomass of rice and wheat. Among PGPR strains tested, Pseudomonas 96–51 and its extract containing growth hormones increased root area, root length and plant biomass of rice and wheat.

Kinetics of high-Level of ß-glucosidase production by a 2-deoxyglucose-resistant mutant of Humicola lanuginosa in submerged fermentation

Um mutante de Hemicola lanuginosa resistente a 2-deoxiglucose(M7) foi obtido atraves de exposicao de conidios a raios γ, permitindo a expressao em caldo contendo 0,6% de 2-deoxiglucose (DG) e celobiose (1%) antes da semeadura em agar DG esculina citrato de ferro amoniacal, da qual foram selecionadas as colonias com halo negro. Os parâmetros cineticos para producao aumentada de s-glucosidase (BGL) foram obtidos empregando-se sabugo de milho como fonte de carbono. A combinacao de espiga de milho com agua de maceracao de milho foi a que forneceu os valores mais altos nos parâmetros cineticos de formacao de todos os produtos. O efeito da temperatura na cinetica e atributos termodinâmicos da producao de BGL pelas cepas selvagem e M7 foi avaliado empregando-se processo de batelada em oito temperaturas diferentes in frascos em agitacao. O melhor desempenho foi observado a 45oC e 20g.l-1 de espiga de milho em 64h. Tanto a multiplicacao quanto a formacao do produto foram muito altas a 45oC e ambas estavam ligadas em condicoes otimas de trabalho. O rendimento de BGL produzido pelo mutante M7 (1556 U.g-1 de espiga seca) foi significativamente superior aos valores reportados para todos os sistemas fungicos e bacterianos. A mutacao influenciou a termoestabilizacao no microrganismo, sendo que o mutante necessitou de energia de ativacao mais baixa para multiplicacao e valores mais baixos de entalpia e entropia para a formacao do produto quando comparado a cepa selvagem e a outros microrganismos mesofilicos e termotolerantes. Na fase de inativacao, os microrganismos necessitaram valores mais baixos de energia de ativacao, entalpia e entropia para o equilibrio da formacao de produto, confirmando a natureza termofilica da maquina metabolica do mutante.

Novel thermodynamics of xylanase formation by a 2-deoxy-D-glucose resistant mutant of Thermomyces lanuginosus and its xylanase potential for biobleachability.

Production of xylanases by Thermomyces lanuginosus wild type and its deoxyglucose resistant mutant M7 on different substrates was investigated. The mutation conferred catabolite repression resistance as M7 supported xylanase production on glucose-based medium that was 8.8-fold higher than that of wild type. Product formation parameters were highest in media containing a combination of corncob and corn steep liquor. Among the soluble substrates, xylose was the best inducer. In the presence of glucose, the wild type produced 26 IU of xylanase per g of glucose while the xylanase yield of the mutant was 224 IU per g of glucose. Thermodynamic studies showed that M7 required lower Gibbs free energy, enthalpy and entropy for product formation than the wild type. In biobleaching studies an 18.6% decrease in kappa number and 2.63% increase in brightness for enzyme-treated pulp was observed. Moreover prebleaching with M7-derived enzyme resulted in a 27.3% reduction in chlorine demand as compared with that of 18.5% decrease when wild organism-derived enzyme was employed. These improvements indicate that the mutant-derived enzymes possessed a useful prebleaching potential and could be exploited for large-scale application.

Influence of carbon and nitrogen sources and temperature on hyperproduction of a thermotolerant β-glucosidase from synthetic medium by Kluyveromyces marxianus

The effect of carbon source and its concentration, inoculum size, yeast extract concentration, nitrogen source, pH of the fermentation medium, and fermentation temperature on β-glucosidase production by Kluyveromyces marxianus in shake-flask culture was investigated. These were the independent variables that directly regulated the specific growth and β-glucosidase production rate. The highest product yield, specific product yield, and productivity of β-glucosidase occurred in the medium (pH 5.5) inoculated with 10% (v/v) inoculum of the culture. Cellobiose (20 g/L) significantly improved β-glucosidase production measured as product yield (YP/S) and volumetric productivity (QP) followed by sucrose, lactose, and xylose. The highest levels of productivity (144 IU/[L·h]) of β-glucosidase occurred on cellobiose in the presence of CSL at 35°C and are significantly higher than the values reported by other researchers on almost all other organisms. The thermodynamics and kinetics of β-glucosidase production and its deactivation are also reported. The enzyme was substantially stable at 60°C and may find application in some industrial processes.

Optimized expression of a thermostable xylanase 11 A gene from Chaetomium thermophilum NIBGE 1 in Escherichia coli.

The xylanase (Xyn11A) gene (883 bp) was amplified using C. thermophilum DNA as template and cloned into pET-32a (+) and expressed in E. coli BL21 under T(7) promoter. The recombinant xylanase on SDS-PAGE had a molecular mass of 30 kDa. Productivity profiles of xylanase in E. coli recombinant are more than 4-fold of that produced from T. reesei RUTC-30, 5-fold of that produced by the donor and significantly higher than the values reported on other E. coli, and Saccharomyces cerevisiae recombinants. Temperature stability, pH stability, and other kinetic parameters confirmed that the gene product was thermo-stable in alkaline buffer favoring its suitability to bio-bleaching of kraft pulp.

Heterologous Expression of the Antifungal β-chitin Binding Protein CBP24 from Bacillus thuringiensis and its Synergistic Action with Bacterial Chitinases

The genome sequence analysis of Bacillus thuringiensis serovar konkukian S4 has shown to contain two chitinases (Chi74, Chi39) and two chitin-binding proteins (CBP50 and CBP24). The Chi74, Chi39 and CBP50 have been characterized previously. The chitin-binding protein CBP24 was cloned and heterologously expressed in Escherichia coli. The recombinant protein was purified using a Ni-NTA purification system. The purified protein was used to study its substrate binding activity using crystalline chitin variants as substrates. The Bmax and Kd values have shown that it preferably binds to β-type of the crystalline chitin at a range of pH with peak activity between 5.5-7.5. To elucidate the role of CBP24 in the chitin degradation system of S4, the purified chitinases Chi74, Chi39 along with the ChiA from Serratia proteamcualans were used in different combinations with the CBP24 and chitinolytic activity was assayed. It was shown that the CBP24 acts synergistically with chitin degradation activity of bacterial chitinases non-specifically. Moreover, the CBP24 has shown antifungal activity against plant pathogenic fungi Fusarium oxysporum and Rhizoctonia solani. The present study will lead us to develop a technology for environmental friendly conversion of chitin to valuable products.