Muhammad Ibrahim Rajoka

Kinetics and thermodynamics of ethanol production by a thermotolerant mutant of Saccharomyces cerevisiae in a microprocessor‐controlled bioreactor

Aims:  The present investigation deals with the development of thermotolerant mutant strain of yeast for studying enhanced productivity of ethanol from molasses in a fully controlled bioreactor.

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 βcellobiohydrolase (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. aCellulose was the most effective inducer of βcellobiohydrlase 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 α-cellulose containing 8-fold sodium nitrate concentration, maximum volumetric productivities (Qp) of β-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.

Optimization of process variables for minimization of byproduct formation during fermentation of blackstrap molasses to ethanol at industrial scale.

Aims:  To investigate the effect of molasses concentration, initial pH of molasses medium, and inoculum’s size to maximize ethanol and minimize methanol, fusel alcohols, acetic acid and aldehydes in the fermentation mash in industrial fermentors.

Activity and thermostability of carboxymethylcellulase from Aspergillus niger is strongly influenced by noncovalently attached polysaccharides

Removal of non-covalently attached polysaccharides from carboxymethylcellulase (CMCase) of Aspergillus niger improved its activity but decreased its thermostability and protease resistance. The activation energy profile of the hydrolysis of carboxymethylcellulose (CMC) was triphasic with increasing values of 17,-55 and-562 kJ/mol for polysaccharide-free and 19, -21 and -207 kJ/mol for polysaccharide-complexed CMCase. The specificity constant (Vmax/Km) of polysaccharide-free CMCase was 1.41 compared to polysaccharide-complexed CMCase which was only 0.68. The polysaccharide free CMCase had lower thermostability (‘melting point’ = 82°C) and higher protease susceptibility compared to polysaccharide-complexed CMCase (‘melting point’>100°C).

Kinetic analysis of the active site of an intracellular β-glucosidase fromCellulomonas biazotea

Purified β-glucosidase fromCellulomonas biazotea had an apparentKm andV for 2-nitrophenyl β-d-glucopyranoside (oNPG) of 0.416 mmol/L and 0.22 U/mg protein, respectively. The activation energy for the hydrolysis of pNPG of β-glucosidase was 65 kJ/mol. The inhibition by Mn2+vs. oNPG of parental β-glucosidase was of mixed type with apparent inhibition constants of 0.19 and 0.60 µmol/L for the enzyme and enzyme-substrate complex, respectively. Ethanol at lower concentrations activated while at higher concentrations it inhibited the enzyme. The determination of apparent pKa’s at different temperatures and in the presence of 30 % dioxane indicated two carboxyl groups which control theV value. The thermal stability of β-glucosidase decreased in the presence of 10 % ethanol. The half-life of β-glucosidase in 1.75 mol/L urea at 35 °C was 145 min, as determined by 0–9 mol/L transverse urea gradient-PAGE.

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.

Cloning and expression of β-glucosidase genes inEscherichia coli andSaccharomyces cerevisiae using shuttle vector pYES 2.0

Genes for β-glucosidase (Bgl) isolated from a genomic library of the cellulolytic bacterium,Cellulomonas biazotea, were cloned in pUC18 in itsSacI cloning site and transformed toE. coli. Ten putative recombinants showed blackening zones on esculin plates, yellow zones on pNPG plates, in liquid culture and on native polyacrylamide gel electrophoresis activity gels. They fell into three distinct groups. Three representativeE. coli clones carried recombinant plasmids designated pRM54, pRM1 and pRM17. The genes were located on 5.6-, 3.7- and 1.84-kb fragments, respectively. Their location was obtained by deletion analysis which revealed that 5.5, 3.2, and 1.8 kb fragments were essential to code for BglA, BglB, and BglC, respectively, and conferred intracellular production of β-glucosidase onE. coli. Expression of thebgl genes resulted in overproduction of β-glucosidase in the three clones. Secretion occurred into the periplasmic fractions. Three inserts carryingbgl genes from the representative recombinantE. coli were isolated withSacI ligated in the shuttle vector pYES2.0 in itsSacI site and transformed toE. coli andS. cerevisiae. The recombinant plasmids were redesignated pRPG1, pRPG2 and pRPG3 coding for BglA1, BglB1 and BglC1. The cloned genes conferred extracellular production of β-glucosidase onS. cerevisiae and enabled it to grow on cellobiose and salicin. Thegall promoter of shuttle vector pYES2.0 enabled the organisms to produce twice more β-glucosidase than that supported by thelacZ-promoter of pUC18 plasmid inE. coli. The cloned gene can be used as a selection marker for introducing recombinant plasmids in wild strains ofS. cerevisiae The enzyme produced bybgl+ yeast andE. coli recombinants resembles that of the donor with respect to temperature and pH requirement for maximum activity. Other enzyme properties of the β-glucosidases fromS. cerevisiae were substantially the same as those fromC. biazotea.

γ-ray induced mutagenesis ofCellulomonas biazotea for improved production of cellulases

A rifampin-resistant mutant ofCellulomonas biazotea secreted elevated levels of cellulasesin vivo. The cellulase production in the mutant was not inhibited in the presence of 5% glucose, cellobiose or glycerol in the solid medium. The mutant exhibited approximately two- to three-fold enhanced product yields and productivity of cellular β-glucosidase over the wild parent in shake-flask culture studies when grown on either cellulosic or lignocellulosic substrates. Extracellular production of filter paper cellulase (FPase) and endo-glucanase (CMCase) were also significantly (p≤0.05) altered. During growth of the mutant on α-cellulose, the maximum volumetric productivities for CMCase, FPase and β-glucosidase were 52, 23.3, and 15.2 IUL−1 h−1,i.e 118, 121, and 229% their respective values for the parental strain. Some enzyme properties of the mutant cellulases were altered. Mutant-derived cellulases produced higher yields of glucose arising by degradation of bagasse, wheat straw, and α-cellulose (1.53-, 1.57-, and 1.75-fold, respectively).

Thermal Characterization of Purified Glucose Oxidase from A Newly Isolated Aspergillus Niger UAF-1

An intracellular glucose oxidase was isolated from the mycelium extract of a locally isolated strain of Aspergillus niger UAF-1. The enzyme was purified to a yield of 28.43% and specific activity of 135 U mg−1 through ammonium sulfate precipitation, anion exchange and gel filtration chromatography. The enzyme showed high affinity for D-glucose with a Km value of 2.56 mM. The enzyme exhibited optimum catalytic activity at pH 5.5. Temperature optimum for glucose oxidase, catalyzed D-glucose oxidation was 40°C. The enzyme showed a high thermostability having a half-life 30 min, enthalpy of denaturation 99.66 kJ mol−1 and free energy of denaturation 103.63 kJ mol−1. These characteristics suggest the use of glucose oxidase from Aspergillus niger UAF-1 as an analytical reagent and in the design of biosensors for clinical, biochemical and diagnostic assays.

Kinetics of enhanced ethanol productivity using raw starch hydrolyzing glucoamylase from Aspergillus niger mutant produced in solid state fermentation

Aims:  The present investigation deals with the effect of raw starch hydrolyzing glucoamylase by a derepressed mutant of Aspergillus niger on enhanced productivity of ethanol from uncooked starch under non‐aseptic conditions.