The Sickle ß-Thalassemia Phenotype.

Sß-thalassemia (Sß-thal) is common among Gulf Arab patients with sickle cell disease, but the phenotype of this group had not been well-documented. We have studied a group of Kuwaiti patients and compared the phenotype in the homozygotes (SS) and Sß-thal patients. Complete blood count, hemoglobin quantitation, serum bilirubin, and lactate dehydrogenase were determined with standard techniques. The patients were screened for α-globin genotype. The Sß-thal patients were also screened for the HBG2 Xmn-1 polymorphism. ß-Thal mutations were determined by arrayed primer extension or direct sequencing. There were 70 SS and 32 Sß-thal patients with mean ages of 14.8±5.9 and 14.2±5.9 years, respectively. The Sß-thal patients had more frequent, severe pain episodes per year compared with the SS, while the patterns among Sß-thal and Sß-thal patients were not significantly different. There were no differences in the frequencies of acute chest syndrome, gallstones, and blood transfusion in the SS and Sß-thal patients. However, none of the Sß-thal patients had been transfused. Among the Sß-thal patients, 25 had ß-thal and 7 had ß-thal mutations, the most common being cd39 (C→T) and IVS-I-110 (G→A), respectively. Sß-thal shows a severe phenotype in Kuwait, even among those with Sß-thal, in whom the IVS-I-110 (G→A) mutation is predominant.

Clinical and Molecular Characteristics of Non-Transfusion-Dependent Thalassemia in Kuwait.

Although not regularly transfused, patients with non-transfusion-dependent thalassemia (NTDT) are prone to iron overload and its complications. Their molecular, phenotypical and laboratory characteristics vary in different populations and there is a need to document local prevailing patterns. We have reviewed the records of our patients with NTDT in Kuwait and documented their clinical and molecular characteristics in addition to iron status [serum ferritin and liver magnetic resonance imaging (MRI) T2*], management and complications. There were 41 patients, made up of 20 with ß-thalassemia intermedia (ß-TI), 18 with Hb H (ß4) disease and three with Hb E (HBB: c.79G > A)-ß-thalassemia (Hb E-ß-thal); their ages ranged from 3 to 36 years (mean 12.5 ± 7.7). While 18 (43.9%) had been transfused at least once, only three (7.3%) had been transfused on multiple occasions. Three patients had serum ferritin >500 ng/mL; while four of 38 had mild or moderate liver iron overload. Seven (35.0%) of the ß-TI patients were managed with hydroxyurea (HU) with good response. Other complications included five patients with gallstones and one each of hypothyroidism and moyamoya. The most common mutations among the ß-TI patients were IVS-II-1 (G > A) and IVS-I-6 (T > C), while among the Hb H patients, the Saudi α2-globin gene polyadenylation (polyA) (AATAAA > AATAAG) mutation was responsible for all cases either as homozygotes (61.1%) or compound heterozygotes with the α-thal-2 (-α(3.7)) allele (33.3%). Although the pattern of NTDT in Kuwaiti patients is generally mild, there is a need to follow them to adulthood as the complications are cumulative and more prevalent in this group.

Ligase-independent cloning of amylase gene from a local Bacillus subtilis isolate and biochemical characterization of the purified enzyme.

Five hundred ninety-seven bacterial isolates from Turkish hot spring water sources were screened for their ability to produce extracellular α-amylase. Among them, a high enzyme-producing Bacillus subtilis isolate, A28, was selected, and its α-amylase gene was cloned and expressed in Escherichia coli by a ligase-independent method. α-Amylase from the recombinant strain was purified to homogeneity by Q-Sepharose anion exchange and Sephacryl S-100 gel filtration chromatographies. The final yield of the enzyme was about 22.5 % of the initial activity, with a 16.4-fold increase in specific activity compared with the culture lysate. The optimum temperature and pH of the enzyme were 70 °C and 6.0, respectively. The enzyme was highly active at acidic-neutral pH range of 4.5-7.0. The amy28 α-amylase retained 100 % of its activity after incubation at 50 °C for 90 min. Co(+2), Cu(2+), Fe(2+), Fe(3+), Ni(+2), and Zn(+2) caused significant inhibition in enzyme activity, which was not affected by Na(+), Mg(2+), Li(+), and Ba(2+). The activity was inhibited about 70 % upon treatment of the enzyme with 10 mM ethylenediaminetetraacetic acid. However, Ca(2+) ions known as high temperature stabilizer for other amylases did not stimulate the activity of the enzyme. Due to pH stability and thermostability of the recombinant amylase, this enzyme may be suitable in starch processing, brewing, and food industries.

Improved activity and thermostability of Bacillus pumilus lipase by directed evolution.

To improve enzymatic activity of Bacillus pumilus lipases, DNA shuffling was applied to two lipase genes from local B. pumilus isolates. Using a high-throughput activity assay, the mutant with highest activity was selected. This chimeric mutant (L3-3), carrying two crossover positions and three point mutations, has a specific activity 6.4 and 8.2 times higher than the two parent enzymes. The mutant also is more tolerant to various detergents and organic solvents, and has a 9 times longer half-life at 50 °C. Homology modeling of mutant L3-3, based on the highly homologous B. subtilis lipase A, shows that the increased thermostability is likely due to structural rigidification and reduced surface hydrophobicity. Increased specific activity may result from the location of mutations close to the active site. Together, our results show that it is possible to evolve, by DNA shuffling, B. pumilus lipase variants with improved applicability as biocatalysts, even if the two parent enzymes are highly similar.