Extracts from ‘kesinai’ (Streblus asper) leaves were investigated as a potential source of enzymes that can serve as an alternative to calf rennet in cheese making. Different types of extraction buffers were investigated namely sodium acetate buffer (pH 4.2-5.0), phosphate buffer (pH 6.0-7.0) and Tris-HCl buffer (pH 7.0-9.0). Finally, the milk-clotting enzyme was extracted using 100 mM Tris-HCl buffer (pH 7.4) with and without 5.0 mg/mL polyvinylpyrrolidone, 0.015 mL/mL Triton X-100 and 2 mM sodium metabisulphite. Purification was carried out using acetone precipitation, and ion-exchange and size-exclusion chromatographic techniques. Results showed that 100 mM Tris-HCl buffer (pH 7.4) was the most efficient extraction buffer among the buffers used in the extraction study. After the final purification step of size-exclusion chromatography, the enzyme was purified 3.3-fold with 42.3% of recovery. The enzyme showed an optimum temperature and pH at 60°C and pH 7.4, respectively. The enzyme was stable up to 70°C for one hour and the partially purified enzyme retained 83% and 96% of its original activity at pH 6.0 and 8.0, respectively. The molecular weight of the partially enzyme was estimated to be 75.8 kDa on SDS-PAGE. The milk-clotting activity of ‘kesinai’ enzyme was found to be lower than that of commercial Mucor rennet.
This study targeted two candidate genes from the best known regulator of blood pressure; the rennin angiotensin system; the ACE gene I/D polymorphism and the angiotensinogen M235T polymorphism. The study aimed to determine the genotypes trend between two different populations; the primary hypertensive patients, and the normal populations. 126 subjects were involved in this study (86 primary hypertensive patients and 40 normal individuals). All demographic factors were considered and analyzed. Insertion/deletion polymorphisms of the ACE gene were determined by an assay based on the polymerase chain reaction (PCR). Polymorphism analysis using PCR-RFLP procedure was used to identify the missense mutation M235T of the AGT gene. All significant data was collected using standardized case report form. The association of the different genotypes and the subjects’ condition was analyzed using the chi squared and odds ratio analyses. In the pooled analysis of both groups, it was shown that the polymorphisms in these genes were significantly associated with the incidence of primary hypertension, p<0.05. Results also showed that the D allele of the ACE gene may be associated with increased risk of primary hypertension (p<0.05, O.R: 3.0 [C.I: 1.25 – 5.35]). The angiotensinogen M235T polymorphism also showed a significant result; the T allele is associated with increased risk of primary hypertension (p<0.05, O.R: 2.56[C.I: 1.55 – 5.28]). This knowledge of the candidate genes of rennin angiotensin system has rendered it possible to show that gene polymorphism in symphony leads to the individual risk of primary hypertension.
Keywords: ACE, M235T, rennin, hypertension
Study site: University Kebangsaan Malaysia Medical Center and International Medical University Cardiology Clinic