Muhammad Nauman Aftab

Purification and characterization of cloned alkaline protease gene of Geobacillus stearothermophilus

Thermostable alkaline serine protease gene of Geobacillus stearothermophilus B‐1172 was cloned and expressed in Escherichia coli BL21 (DE3) using pET‐22b(+), as an expression vector. The growth conditions were optimized for maximal production of the protease using variable fermentation parameters, i.e., pH, temperature, and addition of an inducer. Protease, thus produced, was purified by ammonium sulfate precipitation followed by ion exchange chromatography with 13.7‐fold purification, with specific activity of 97.5 U mg−1, and a recovery of 23.6%. Molecular weight of the purified protease, 39 kDa, was determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). The enzyme was stable at 90 °C at pH 9. The enzyme activity was steady in the presence of EDTA indicating that the protease was not a metalloprotease. No significant change in the activity of protease after addition of various metal ions further strengthened this fact. However, an addition of 1% Triton X‐100 or SDS surfactants constrained the enzyme specific activity to 34 and 19%, respectively. Among organic solvents, an addition of 1‐butanol (20%) augmented the enzyme activity by 29% of the original activity. With casein as a substrate, the enzyme activity under optimized conditions was found to be 73.8 U mg−1. The effect of protease expression on the host cells growth was also studied and found to negatively affect E. coli cells to certain extent. Catalytic domains of serine proteases from eight important thermostable organisms were analyzed through WebLogo and found to be conserved in all serine protease sequences suggesting that protease of G. stearothermophilus could be beneficially used as a biocontrol agent and in many industries including detergent industry.

Systematic mutagenesis method for enhanced production of bacitracin by Bacillus licheniformis mutant strain UV-MN-HN-6

The purpose of the current study was intended to obtain the enhanced production of bacitracin by Bacillus licheniformis through random mutagenesis and optimization of various parameters. Several isolates of Bacillus licheniformis were isolated from local habitat and isolate designated as GP-35 produced maximum bacitracin production (14±0.72 IU ml-1). Bacitracin production of Bacillus licheniformis GP-35 was increased to 23±0.69 IU ml-1 after treatment with ultraviolet (UV) radiations. Similarly, treatment of vegetative cells of GP-35 with chemicals like N-methyl N’-nitro N-nitroso guanidine (MNNG) and Nitrous acid (HNO2) increased the bacitracin production to a level of 31±1.35 IU ml-1 and 27±0.89 IU ml-1respectively. Treatment of isolate GP-35 with combined effect of UV and chemical treatment yield significantly higher titers of bacitracin with maximum bacitracin production of 41.6±0.92 IU ml-1. Production of bacitracin was further enhanced (59.1±1.35 IU ml-1) by optimization of different parameters like phosphate sources, organic acids as well as temperature and pH. An increase of 4.22 fold in the production of bacitracin after mutagenesis and optimization of various parameters was achieved in comparison to wild type. Mutant strain was highly stable and produced consistent yield of bacitracin even after 15 generations. On the basis of kinetic variables, notably Yp/s (IU/g substrate), Yp/x (IU/g cells), Yx/s(g/g), Yp/s, mutant strain B. licheniformis UV-MN-HN-6 was found to be a hyperproducer of bacitracin.

Optimization of saccharification potential of recombinant xylanase from Bacillus licheniformis

ABSTRACT Saccharification potential of xylanase enzyme cloned from Bacillus licheniformis into E. coli BL21 (DE3) was evaluated against plant biomass for the production of bioethanol. The expression of cloned gene was studied and conditions were optimized for its large scale production. The parameters effecting enzyme production were examined in a fermenter. Recombinant xylanase has the ability to breakdown birchwood xylan to release xylose as well as the potential to treat plant biomass, such as wheat straw, rice straw, and sugarcane bagass. The saccharification ability of this enzyme was optimized by studying various parameters. The maximum saccharification percentage (84%) was achieved when 20 units of recombinant xylanase were used with 8% sugarcane bagass at 50°C and 120 rpm after 6 hours of incubation. The results indicated that the bioconversion of natural biomass by recombinant xylanase into simple sugars can be used for biofuel (bioethanol) production. This process can replace the use of fossil fuels, and the use of bioethanol can significantly reduce the emission of toxic gases. Future directions regarding pre-treatment of cellulosic and hemicellulosic biomass and other processes that can reduce the cost and enhance the yield of biofuels are briefly discussed.

Expression of thermostable β-xylosidase in Escherichia coli for use in saccharification of plant biomass

ABSTRACT The present work is aimed to evaluate the saccharification potential of a thermostable β-xylosidase cloned from Bacillus licheniformis into Escherichia coli for production of bioethanol from plant biomass. Recombinant β-xylosidase enzyme possesses the ability of bioconversion of plant biomass like wheat straw, rice straw and sugarcane bagass. By using this approach, plant biomass that mainly constitute cellulose can be converted to reducing sugars that could then be easily converted to bioethanol by simple fermentation process. The production of bioethanol will help to overcome energy requirements due to depleting fossil fuels and will also help to protect environment by reducing greenhouse gas emission. In the end, future directions are briefly mentioned that can be utilized to reduce the cost and increase the yield of biofuels.

Cloning, purification, and characterization of xylose isomerase from Thermotoga naphthophila RKU-10.

A 1.3 kb xyl‐A gene encoding xylose isomerase from a hyperthermophilic eubacterium Thermotoga naphthophila RKU‐10 (TnapXI) was cloned and over‐expressed in Escherichia coli to produce the enzyme in mesophilic conditions that work at high temperature. The enzyme was concentrated by lyophilization and purified by heat treatment, fractional precipitation, and UNOsphere Q anion‐exchange column chromatography to homogeneity level. The apparent molecular mass was estimated by SDS–PAGE to be 49.5 kDa. The active enzyme showed a clear zone on Native‐PAGE when stained with 2, 3, 5‐triphenyltetrazolium chloride. The optimum temperature and pH for D‐glucose to D‐fructose isomerization were 98 °C and 7.0, respectively. Xylose isomerase retains 85% of its activity at 50 °C (t1/2 1732 min) for 4 h and 32.5% at 90 °C (t1/2 58 min) for 2 h. It retains 90–95% of its activity at pH 6.5–7.5 for 30 min. The enzyme was highly activated (350%) with the addition of 0.5 mM Co2+ and to a lesser extent about 180 and 80% with the addition of 5 and 10 mM Mn2+ and Mg2+, respectively but it was inhibited (54–90%) in the presence of 0.5–10 mM Ca2+ with respect to apo‐enzyme. D‐glucose isomerization product was also analyzed by Thin Layer Chromatography (Rf 0.65). The enzyme was very stable at neutral pH and sufficiently high temperature and required only a trace amount of Co2+ for its optimal activity and stability. Overall, 52.2% conversion of D‐glucose to D‐fructose was achieved by TnapXI. Thus, it has a great potential for industrial applications.