Kinetics and Regulation Studies of the Production of β-Galactosidase from Kluyveromyces marxianus Grown on Different Substrates

Lactose-intolerance is manifested in 50 % of the world’s population. This can be remediated by removing lactose from the diet or converting it into glucose and galactose with β-galactosidase (EC 3.2.1.23). In this work, batch production of this enzyme in the presence of lactose, galactose, cellobiose, xylose, arabinose, sucrose and glucose was investigated using Kluyveromyces marxianus in shake flask culture studies. Substrate type and temperature were the independent variables that directly regulated the specific growth and β-galactosidase production rates. Lactose (2 %) supported the maximum specific product yield (YP/X), followed by galactose, sucrose, cellobiose, xylose, arabinose and glucose. Its synthesis was regulated by an induction and a growth-dependent repression mechanism. The optimum temperature for the production was found to be 35–37 °C. The highest volumetric productivity of enzyme (80.0 IU/L/h) occurred on lactose-corn steep liquor medium. This was significantly higher than the calculated values reported in the literature. Thermodynamic studies revealed that the cells provided a defence mechanism against thermal inactivation. The enzyme was stable at 60 °C and pH=5.0–7.0, and it may find application in commercial lactose hydrolysis

Enhanced sporulation and toxin production by a mutant derivative of Bacillus thuringiensis

Bacillus thuringiensis (Bt) strains along with insecticidal crystal proteins (ICP) also produce proteases, and chitinases during growth and sporulation. These both potentate the activity of ICPs. This work aimed to obtain bioinsecticide over-production and thermotolerant mutant through classical mutagenesis of vegetative cells of Bt by using UV-mutation multiple times. The isolated survivors were screened on the basis of their production of β-glucosidase, delta-endotoxins and biomass in glucose-based medium. Maximum spore and crystal proteins were produced at 40°C with corn steep liquor as nitrogen source and hydrol as a carbon source. The best mutant MUV7 supported significantly (P = 0.0001) higher values of all kinetic parameters than those supported by the wild culture. Lower demand of activation energy (47.4 kJ mol-1) for crystal protein formation revealed that the best mutant was comparable with those of thermophilic group of organisms.Key words: Bioinsecticide, biomass, delta-endotoxin, insecticidal crystal proteins, kinetics, spores

DNA damage and plasma homocysteine levels are associated with serum metabolites and mineral constituents’ profiles in children with persistent diarrhea

This study describes the association between levels of DNA damage and homocysteine (Hcy) in persistent diarrheic (PD) patients and correlates them with serum biochemical metabolites and mineral components. PD patients (n = 36) age 4 - 6 years from Faisalabad hospitals were examined for anthropometric factors, plasma biochemical and mineral constituents. Compared to 36 normal controls, children with PD had significantly higher concentrations of LDL (p = 0.0001), ALT (p = 0.01),homocysteine (p = 0.001), TOS (p = 0.0001), TBARS (p = 0.001), K (p = 0.0001) and Mg (p = 0.0001) while serum triglyceride, total proteins, albumin, globulin, T3, T4, TAS, Na, Ca, Zn and Cu were significantlylower than those of healthy individuals. Both DNA damage and Hcy were positively linked with LDLcholesterol, TBARS and K (all p values < 0.05). Both Hcy profile and percentage DNA damage in PD patients may impart role in the endothelium damage even in the normal range. PD patients have severe deficiency of macro- and micro-nutrients which may have resulted in enhancement of oxidative stress, DNA damage and Hcy levels in patients’ plasma. Appropriate supplementation of macro- and micronutrients may decrease the DNA damage, Hcy levels and enhance the levels of health markers and decrease the mortality rate of PD patients

Biolistic transformation of Saccharomyces cerevisiae with β-glucosidase gene from Cellulomonas biazotea

A β-glucosidase genomic DNA from Cellulomonas biazotea NIAB 442 was isolated and coated onto tungsten microprojectiles for direct transformation of the gene into Saccharomyces cerevisiae. Transformation of β-glucosidase into S. cerevisae conferred the ability to hydrolyse esculin and cellobiose, indicated that the gene is expressed in the bombarded yeast. Key Words: Biolistic transformation, β-glucosidase, Cellulomonas biazotea, Saccharomyces cerevisiae. African Journal of Biotechnology Vol.3(1) 2004: 112-11

Influence of various fermentation variables on exo-glucanase production in Cellulomonas flavigena

The influence of carbon and nitrogen sources on the production of exo-glucanase was investigated. The enzyme production was variable according to the carbon or nitrogen source used. Levels of \u3b2-cellobiohydrolase (CBH) were minimal in the presence of even low concentrations of glucose. Enzyme production was stimulated by other carbohydrates and thus is subject to carbon source control by easily metabolizable sugars. In Dubos medium, on cellobiose, the cellobiohydrolase titres were 2-to 110-fold higher with cells growing on monomeric sugars and 2.7 times higher than cells growing on other disaccharides. \u3b1-Cellulose was the most effective inducer of \u3b2-cellobiohydrlase and filter paperase (FPase) activities, followed by kallar grass straw. Exogenously supplied glucose inhibited the synthesis of the enzyme in cultures of Cellulomonas flavigena . Nitrates were the best nitrogen sources and supported greater cell mass, cellobiohydrolase and FPase production. During growth on \u3b1-cellulose containing 8-fold sodium nitrate concentration, maximum volumetric productivities (Qp) of \u3b2-cellobiohydrolase and FPase were 87.5 and 79.5 IU/l./h respectively and are significantly higher than the values reported for some other potent fungi and bacteria

Enhanced production of subtilisin of Pyrococcus furiosus expressed in Escherichia coli using autoinducing medium

A subtilisin gene identified in the reported genome sequence of Pyrococcus furiosus was amplified and inserted in pET-22b(+) vector to produce the recombinant plasmid pET-SB. Escherichia coli BL-21 (DE3)CodonPlus was transformed with this plasmid and the enzyme was expressed up to 30% of the total cell protein on induction with IPTG. The expressed protein appeared at a position corresponding to ~20 kDaon SDS-PAGE as compared to theoretical molecular mass of 17.6 kDa. This aberrant electrophoresis mobility could be due to specific amino acid composition of the protein. Auto-induction with lactose also produced a similar level of expression but the total amount of the enzyme produced was 2.4 foldgreater than that when produced with IPTG induction. This was due to a higher cell density obtainable in the auto-inducing medium. The enzyme expressed in the insoluble state could be partially refolded after denaturation with urea at high pH. This study reports for the first time high-level expression ofsubtilisin of P. furiosus in E. coli using an auto-inducing medium

Hyper-production of a thermotolerant \u3b2-xylosidase by a deoxy-D-glucose and cycloheximide resistant mutant derivative of Kluyveromyces marxianus PPY 125

Production of \u3b2-xylosidaseby a cycloheximide and 2-deoxy-D-glucose-resistant mutant of Kluyveromyces marxianus PPY125 was studied when cultured on growth media containing galactose, glucose, xylose, cellobiose, sucrose and lactose as carbon sources. Xylose, cellobiose, lactose and sucrose were the key substrates. Both K. marxianus PPY125 and its mutant (M 125) supported maximum \u3b2-xylosidase specific product yield (YP/X) following growth on xylose. Basal level of activity was observed in non-induced cultures grown on glucose. The mutant produced 1.5 to 2-fold more \u3b2-xylosidase than that produced by the wild cells. Synthesis of \u3b2-xylosidase was regulated by an induction mechanism in both wild and mutant cells. Addition of glucose did not inhibit the synthesis of \u3b2-xylosidase in both parental and mutant cultures in the presence of corn steep liquor. Partially purified enzyme showed good stability when incubated at 60\ub0C and was quite stable at pH 5.0-7.0. Thermodynamic studies revealed that the enzyme derived by the mutant M125 was more thermostable as evidenced by higher midpoint inactivation temperature, lower activation energy demand for \u3b2-xyloside hydrolysis, as well as lower enthalpy and entropy demand for reversible denaturation of enzyme