Escherichia coli glucuronylsynthase: an engineered enzyme for the synthesis of beta-glucuronides.

The glycosynthase derived from E. coli beta-glucuronidase catalyzes the glucuronylation of a range of primary, secondary, and aryl alcohols with moderate to excellent yields. The procedure provides an efficient, stereoselective, and scalable single-step synthesis of beta-glucuronides under mild conditions.

Cloning and characterization of Thermotoga maritima beta-glucuronidase.

The putative beta-glucuronidase from Thermotoga maritima, comprising 563 amino acid residues conjugated with a Hisx6 tag, was cloned and expressed in Escherichia coli. The enzyme has a moderately broad specificity, hydrolysing a range of p-nitrophenyl glycoside substrates, but has greatest activity on p-nitrophenyl beta-D-glucosiduronic acid (kcat=68 s(-1), kcat/K(M)= 4.5x10(5) M(-1) s(-1)). The enzyme also shows a relatively broad pH-dependence with activity from pH4.5 to 7.5 and a maximum at pH6.5. As expected the enzyme is stable towards heat denaturation, with a half life of 3h at 85 degrees C, in contrast to the mesophilic E. coli enzyme, which has a half life of 2.6h at 50 degrees C. The identity of the catalytic nucleophile was confirmed as Glu476 within the sequence VTEFGAD by trapping the glycosyl-enzyme intermediate using the mechanism-based inactivator, 2-deoxy-2-fluoro-beta-D-glucosyluronic acid fluoride and identifying the labeled peptide in peptic digests by HPLC-MS/MS methodologies. Consistent with this, the Glu476Ala mutant was shown to be hydrolytically inactive. The acid/base catalyst was confirmed as Glu383 by generation and kinetic analysis of enzyme mutants modified at that position, Glu383Ala and Glu383Gln. The demonstration of activity rescue by azide is consistent with the proposed role for this residue. This enzyme therefore appears suitable for use in enzymatic oligosaccharide synthesis in either the transglycosylation mode or by use of glycosynthase and thioglycoligase approaches.

The use of a molecular technique for the detection of porcine ingredients in the Malaysian food market.

OBJECTIVE: To develop a molecular technique that is fast and reliable in detecting porcine contamination or ingredients in foods. METHODS: The method applied involved DNA amplification using polymerase chain reaction (PCR) technology. Thus, the sequence of a certain gene found uniquely in pork was identified and its sequence was used to design specific primers for the PCR. The extraction of DNA was optimized in respect to PCR and detection limits were established. The optimized method was then used to identify pork in food products obtained from various local hypermarkets. The latest results were confirmed in triplicates on the 20th April 2006 at the Molecular Biology Laboratory, International Islamic University, Malaysia. RESULTS: The method was shown to be robust and reliable. Out of 30 food samples not expected to contain pork material, 3 samples were shown to be contaminated with pork material; 2 chocolates and one chicken nugget. CONCLUSION: We observed that 2 food products that were labeled as halal showed positive for porcine ingredients, while another that did not have any halal logo but originated from outside Malaysia and exported to many Middle Eastern nations also showed positive.

Recombinant bromelain production in Escherichia coli: process optimization in shake flask culture by response surface methodology.

Bromelain, a cysteine protease with various therapeutic and industrial applications, was expressed in Escherichia coli, BL21-AI clone, under different cultivation conditions (post-induction temperature, L-arabinose concentration and post-induction period). The optimized conditions by response surface methodology using face centered central composite design were 0.2% (w/v) L-arabinose, 8 hr and 25°C. The analysis of variance coupled with larger value of R2 (0.989) showed that the quadratic model used for the prediction was highly significant (p < 0.05). Under the optimized conditions, the model produced bromelain activity of 9.2 U/mg while validation experiments gave bromelain activity of 9.6 ± 0.02 U/mg at 0.15% (w/v) L-arabinose, 8 hr and 27°C. This study had innovatively developed cultivation conditions for better production of recombinant bromelain in shake flask culture.