| D-tagatose, a novel low-calorie sugar-substituting sweetener, possesses multi-functionality applied in food and pharmaceuticals, such as anti-hyperglycemia and probiotics. D-Tagatose is a hexoketose monosaccharide sweetener, which is an isomer of D-galactose and is rarely found in nature.L-Arabinose isomerase (L-AI) is an intracellular enzyme that catalyzes the reversible isomerization of L-arabinose to L-ribulose involved in either the pentose phosphate or the phosphoketolase pathway. Thermophilic L-AIs have been reported to possess a catalytic activity for the conversion of D-galactose to D-tagatose. This bifunctional activity could be exploited industrially for the production of D-tagatose.In this article, cloning and screening of the araA gene from the anaerobic thermophilic bacterium Thermoanaerobacter mathranii, the enzymatic engineering, the immobilization of L-arabinose isomerase, the tool enzyme for tagatose production, and separation and purification of D-tagatose were elaborated.The araA gene encoding L-arabinose isomerase from Thermoanaerobacter mathranii was cloned, sequenced and over-expressed in E. coli BL21(DE3). This gene encodes a496-amino acid protein with a calculated molecular weight of53kDa. The TaMAI was optimally active at pH8.0and60℃. The enzyme activity was obviously improved in presence of5mM Mn2+. The TaMAI converted the D-galactose into D-tagatose with a high conversion rate of39.5%after20h at60℃.In order to increase the production of TaMAI, the fermentation conditions of engineering bacteria E.coli BL21(DE3)(pET-15b-TaMAI) were studied. The optimal induction conditions as follows:LB culture medium,0.5mM IPTG,37℃,4h.Various materials and methods were used to immobilize TaMAI. It has been found that two different methods are with higher relative activity and better using stability. The first is sodium alginate enzyme immobilization, the second is sodium alginate combined with active carbon enzyme immobilization. The effects of pH, temperature on D-tagatose production reaction catalyzed by immobilized TaMAI were investigated. The results showed that the optimum pH of the two immobilized enzymes were both shifed from8.0to7.5. The optimum temperature of the former is75℃, the later is70℃, which is higher than that of the free enzyme. The temperature stability and pH stability of the two immobilized enzymes were better than that of the free enzyme. The two immobilized enzymes also showed good operation stability, the half-life of them were9d and7d, respectively.The continuous enzymatic conversion of D-galactose to D-tagatose with an immobilized TaMAI in packed-bed reactor was studied. The optimum reaction yield with immobilized TaMAI increased by four percentage points to43..9%compared with that of free TaMAI. The highest productivity of10g/L·h was achieved with the yield of23.3%. Continuous production was performed at70℃, after168h the reaction yield was still above30%.Two methods for D-tagatose purification were studied. Firstly, the resultant syrup was incubated with Saccharomyces cerevisiae LI cells. The selectively degradation of D-galactose was achieved to obtain high purity D-tagatose. This method was firstly used in D-tagatose purification all over the world. Secondly, the technology of separation and purification of D-tagatose was established according to the method of DTF-01Ca2+cation exchange chromatography. The optimal Ca2+cation exchange chromatography conditions: column temperature of70℃, the input volume of3mL and the flow velocity of3mL/min. Ion chromatography analysis showed that the purity of D-tagetose achieved by those two methods above reached95%.The subunit structure of L-AIs is organised into three domains:an N-terminal. a central and a C-terminal domain. The active centre of TaMAI located in central and C-terminal domain. In order to clarify the function of the N-terminal domain, we disparity trunctuated the TaMAI in N-terminal by the PCR. The results show that N-terminal domain was crucial for soluble expression and enzyme activity of TaMAI.According to the E.coli K12AI (ECAI) crystal structure analysis, and results about other sources L-AIs directed evolution published, we speculated six important amino acid sites which have a significant influence on enzyme activity and substrate affinity of the TaMAI through the sequence alignment. Site-directed mutagenesis was done in residues Asn223, His313, Ala375, Ser384, G400, Arg419, C441, respectively. Biochemical characterizations of six mutations were well studied. The enzyme activity and thermostability of four mutations were improved. |
PDF Full Text Request