Sucrose phosphorylase (EC 2.4.1.7, Sucrose phosphorylase, SPase) can be produced by recombinant strain Escherichia coli Rosetta(DE3)/Pet-SPase. Crude enzyme was obtained from the cells by the high pressure disruption and centrifugation. Sucrose phosphorylase was purified by Ni-NTA affinity column chromatography and desalted by ultrafiltration. The specific enzyme activity was 1.1-fold higher than that of the crude enzyme, and recovery rate was 82.7%. The purified recombinant SPase had a band of 59 kDa on SDS-PAGE. Thermostability of the enzyme was shown at temperatures up to 37 degrees C, and pH stability between pH 6.0 and 6.7. The optimum temperature and pH were 37 degrees C and 6.7, respectively. The K(m) of SPase for sucrose was 7.3 mmol/L, and Vmax was 0.2 micromol/(min x mg). Besides, alpha-arbutin was synthesized from sucrose and hydroquinone by transglucosylation with recombinant SPase. The optimal conditions for synthesis of alpha-arbutin were 200 U/mL of recombinant SPase, 20% of sucrose, and 1.6% hydroquinone at pH 6-6.5 and 25 degrees C for 21 h. Under these conditions, alpha-arbutin was obtained with a 78.3% molar yield with respect to hydroquinone, and the concentration of alpha-arbutin was about 31 g/L.
Arbutin ; biosynthesis ; Catalysis ; Enzyme Stability ; Escherichia coli ; enzymology ; genetics ; Glucosyltransferases ; biosynthesis ; genetics ; metabolism ; Hydroquinones ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; metabolism ; Sucrose ; metabolism

Objective:To investigate the clinical characteristics of patients complicated with Hashimoto’s thyroiditis (HT) and papillary thyroid micro-carcinoma (PTMC) and risk factors for central lymph node metastasis (CLNM) by analyzing the clinical data.Methods:Clinical data of 770 patients with PTMC admitted to Head and Neck Surgery Center of Sichuan Cancer Hospital from May. 2015 to Nov. 2017 were retrospectively analyzed, including 250 HT-PTMC patients (observation group) and 520 non-HT-PTMC patients (control group) . There were 197 males and 573 females, with a male to female ratio of 1.00:2.91. Into observation indexes included patient’s age, gender, serum thyroid stimulating hormone (TSH) , thyroglobulin antibody (TG-Ab) , thyroid peroxidase antibody (TPO-Ab) , number of foci, diameter of foci, calcification of foci, location of foci (with or without extra-glandular invasion) , number of nodules (no matter benign or malignant) , and lymph node metastasis in central and lateral cervical regions. SPSS 22.0 software was used for statistical analysis. Normally distributed data were expressed ± s. The difference between observation group and control group was compared by chi-square test of single factor analysis. The risk factors of CLNM of the observation group were analyzed with multivariate Logistic regression, the difference was statistically significant if P<0.05. Results:There were statistically significant differences between the observation group and the control group in age ( P=0.006) , gender ( P<0.001) , TSH ( P<0.001) , TG-Ab ( P<0.001) , TPO-Ab ( P<0.001) , number of nodules ( P=0.016) , and central lymph node ( P<0.001) . Compared with non-HT-PTMC group, HT-PTMC group had a higher proportion of women under 55 years old, and both TG-Ab and TPO-Ab had higher positive rates. Patients with HT-PTMC were more likely to show polynodule changes, but their central lymph node metastasis rate was lower than that of the non-HT-PTMC group. Single-factor analysis showed that the number of cancer foci, calcification of cancer foci, and location of cancer foci were significantly correlated with the CLNM of HT-PTMC patients (all P<0.001) . Multivariate Logistic regression analysis showed that multiple cancer foci ( P<0.001) , invasion and capsule ( P<0.001) , and cancer foci with calcification ( P=0.005) were independent risk factors for CLNM. Conclusions:HT-PTMC is more common in women under 55 years of age, and most of them show multiple nodules in bilateral glandular lobes, often accompanied by elevated serum TSH, TG-Ab and TPO-Ab levels. Meanwhile, the lymph node metastasis rate of HT-PTMC is relatively low, and the prognosis may be relatively good. In HT-PTMC, the risk of multiple carcinoma foci, cancer foci with calcification, cancer foci invasion and the occurrence of CLNM is higher. Preventive central lymph node dissection helps to reduce the occurrence of postoperative cervical lymph node recurrence.