The β glucosidase Tm bglA gene from hyperthermophile Thermotoga maritima was cloned into expression vectors pET 28a, producing two proteins of 55 kDa and 52 kDa in cell, which could be referred to Tm BglA with and without 23 amino acids. The results showed that fusion of 23 amino acids to Tm BglA improved its soluble expression and product tolerance, resulting over 60% yield of recombinant Tm BglA secreted into the growth medium in Escherichia coli JM109 (DE3). Tm BglA with 23 amino acids had higher k cat and K M values for p nitrophenyl β D glycopyranoside and much stronger product inhibition than Tm BglA without 23 amino acids. Subsequently, the Tm BglA was immobilized on chitin efficiently by genetically fusing the chitin binding domain of chitinase A1 from Thermoanaerobacterium thermosaccharolyticum DSM571. The immobilized Tm BglA had higher optimal temperature (95°C) and was more thermostable at the range from 75 to 100°C than its free form. This immobilized protein exhibited high stability and the con version yield exceeding 90%. The high level soluble expression, combined simultaneous purification and immobilization of the enzyme on chitin offers a novel approach for the low cost production of β glucosidase to produce lactose free fresh dairy products.

A time-resolved fl uoroimmunoassay (TR-FIA) for determination of clenbuterol (CLB) in pig liver was developed. The limit of detection (LOD) was 0.02 ng g -1 and limit of quantifi cation (LOQ), 0.08 ng g -1 . Recoveries ranged from 89.3 to 117.9% for pig liver at spiked levels of 0.1-5 ng g -1 . The results obtained by the TR-FIA and enzyme-linked immunosorbent assay (ELISA) showed a good correlation. The established TR-FIA was applied for screening pig liver from the local market and confi rmed by gas chromatography-mass spectrometry (GC-MS). This proposed technique could be used for routine screening for drug residues.