Site-directed mutagenesis: role of lid region for T1 lipase specificity.

A broad substrate specificity enzyme that can act on a wide range of substrates would be an asset in industrial application. T1 lipase known to have broad substrate specificity in its native form apparently exhibits the same active sites as polyhydroxylalkanoate (PHA) depolymerase. PhaZ6Pl is one of the PHA depolymerases that can degrade semicrystalline P(3HB). The objective of this study is to enable T1 lipase to degrade semicrystalline P(3HB) similar to PhaZ6Pl while maintaining its native function. A structural study on PhaZ6Pl contains no lid in its structure and therefore T1 lipase was designed with removal of its lid region. BSLA lipase was chosen as the reference protein for T1 lipase modification since it contains no lid. Initially, structures of both enzymes were compared via protein-protein superimposition in 3D-space and the location of the lid region of T1 lipase was highlighted. A total of three variants of T1 lipase without lid were successfully designed by referring to BSLA lipase (a lipase without lid). The ability of T1 lipase without lid variants in degrading P(3HB) was investigated quantitatively. All the variants showed activity towards the substrate which confirmed that T1 lipase without lid is indeed able to degrade P(3HB). In addition, D2 was recorded to have the highest activity amongst other variants. Results obtained in this study highlighted the fact that native T1 lipase is a versatile hydrolase enzyme which does not only record triglyceride degradation but also P(3HB) by simply removing the lid region.

Ability of T1 Lipase to Degrade Amorphous P(3HB): Structural and Functional Study.

An enzyme with broad substrate specificity would be an asset for industrial application. T1 lipase apparently has the same active site residues as polyhydroxyalkanoates (PHA) depolymerase. Sequences of both enzymes were studied and compared, and a conserved lipase box pentapeptide region around the nucleophilic serine was detected. The alignment of 3-D structures for both enzymes showed their active site residues were well aligned with an RMSD value of 1.981 Å despite their sequence similarity of only 53.8%. Docking of T1 lipase with P(3HB) gave forth high binding energy of 5.4 kcal/mol, with the distance of 4.05 Å between serine hydroxyl (OH) group of TI lipase to the carbonyl carbon of the substrate, similar to the native PhaZ7 Pl . This suggests the possible ability of T1 lipase to bind P(3HB) in its active site. The ability of T1 lipase in degrading amorphous P(3HB) was investigated on 0.2% (w/v) P(3HB) plate. Halo zone was observed around the colony containing the enzyme which confirms that T1 lipase is indeed able to degrade amorphous P(3HB). Results obtained in this study highlight the fact that T1 lipase is a versatile hydrolase enzyme which does not only record triglyceride degradation activity but amorphous P(3HB) degradation activity as well.

Rate of seroconversion in repeat blood donors at the national blood centre, kuala lumpur.

The World Health Organization (WHO) recommend that all donated blood are to be screened for at least three viral infections [human immunodeficiency virus (HIV), hepatitis C virus (HCV), hepatitis B virus (HBV)]. The National Blood Centre, Kuala Lumpur (NBCKL) aims to reduce transfusion transmitted infections (TTI) as it still remains as one of the major risk for blood transfusion. A cross sectional study was conducted at the National Blood Centre, Kuala Lumpur from 1st January 2009 to 31st June 2010. Data from 581,020 donors were analyzed from year 2004 to 2008. All data were retrieved from NBCKL Blood Bank Information System (BBIS). A total of 201 repeat donors were included in the study based on the inclusion criteria but only 132 repeat donors agreed to participate. Information on sociodemographic, risk factors, knowledge of donors and high risk behavior were extracted from standardize questionnaire. Data were analyzed using SPSS version 14.0. The aim of this study was to determine the predictors of the seropositive infectivity among repeat blood donors at the NBCKL. The results showed Syphilis accounts for the highest and increasing seroconversion rate among other infections from 20.83 % in year 2004 to 44.6 % in year in year 2008. HIV and HCV infection also showed increasing seroconversion rate in 5 years' time from 6.41 % in year 2004 to 17.54 % in year 2008 and 4.8 % in year 2004 to 5.94 % in year 2008 respectively. However, HBV infection alone showed a decreasing seroconversion rate from 20.83 % in year 2004 to 10.4 % in year 2008. Level of donors' awareness regarding high risk factors (activities or behaviour) can lead to higher risk of TTI with significant p value in this predictors model(p < 0.05). Repeat blood donors with high risk activities are more likely to have seropositive results for HBV, HIV and Syphilis. This study found that the frequency of HCV seropositivity is higher among lapsed donor. Socio demographic factors such as male and working in the private sector predominates in all TTI markers. Majority of the respondents were aware about relation of high risk activities and risk of TTI.

Effects of plateletpheresis on blood coagulation parameters in healthy donors at National Blood Centre, Kuala Lumpur, Malaysia.

Plateletpheresis is a method used to remove platelet from the body either from random volunteer donors, patient's family members or HLA matched donors. A cross sectional study was carried out on 59 plateletpheresis donors aged between 18 and 55 years at National Blood Center (NBC), Kuala Lumpur. We compared the blood parameters before and after plateletpheresis and we found that the platelet count, FVIII, fibrinogen and thrombophilia markers anti-thrombin (AT), protein C and protein S were significantly reduced (p<0.05) with prolonged PT and APTT. There were significant changes in blood coagulation parameters but it is within acceptable range.