Combined strategies for improving production of a thermo-alkali stable laccase in Pichia pastoris

Background: Laccases are copper-containing enzymes which have been used as green biocatalysts for many industrial processes. Although bacterial laccases have high stabilities which facilitate their application under harsh conditions, their activities and production yields are usually very low. In this work, we attempt to use a combinatorial strategy, including site-directed mutagenesis, codon and cultivation optimization, for improving the productivity of a thermo-alkali stable bacterial laccase in Pichia pastoris. Results: A D500G mutant of Bacillus licheniformis LS04 laccase, which was constructed by site-directed mutagenesis, demonstrated 2.1-fold higher activity when expressed in P. pastoris. The D500G variant retained similar catalytic characteristics to the wild-type laccase, and could efficiently decolorize synthetic dyes at alkaline conditions. Various cultivation factors such as medium components, pH and temperature were investigated for their effects on laccase expression. After cultivation optimization, a laccase activity of 347 ± 7 U/L was finally achieved for D500G after 3 d of induction, which was about 9.3 times higher than that of wild-type enzyme. The protein yield under the optimized conditions was about 59 mg/L for D500G. Conclusions: The productivity of the thermo-alkali stable laccase from B. licheniformis expressed in P. pastoris was significantly improved through the combination of site-directed mutagenesis and optimization of the cultivation process. The mutant enzyme retains good stability under high temperature and alkaline conditions, and is a good candidate for industrial application in dye decolorization.

Risk Factors for No-Reflow Phenomenon after Percutaneous Coronary Intervention in Patients with Acute Coronary Syndrome.

BACKGROUND: To explore risk factors for no-reflow phenomenon after percutaneous coronary intervention in patients with acute coronary syndrome. METHODS: A total of 733 acute myocardial infarction patients with persistent ischemic chest pain within 12 or 12-24 hours after onset received emergency percutaneous coronary intervention. Patients were divided into a normal reflow group and a no-reflow group, according to TIMI grading and myocardial blush grading after percutaneous coronary intervention. Related risk factors were analyzed. RESULTS: The incidence of no-reflow phenomenon after percutaneous coronary intervention was 16.1%. Univariate analysis showed that, compared with the normal reflow group, the no-reflow group was older, reperfusion time was significantly longer, preoperative systolic pressure was lower, troponin peak was higher, and creatine kinase enzyme peak was higher (p < 0.05). The proportions of preoperative cardiac function Killip grade ≥ 2 and number of patients using preoperative intra-aortic balloon pump were significantly different (p < 0.05). Multivariate logistic regression analysis showed that age > 65 years (OR: 1.471; 95% CI: 1.462-1.492; p = 0.007), reperfusion time > 6 hours (OR: 1.274; 95% CI: 1.164-1.405; p = 0.001), low systolic pressure at admission (< 100 mmHg) (OR: 1.918; 95% CI: 1.017-3.897; p = 0.004), intra-aortic balloon pump use before percutaneous coronary intervention (OR: 1.949; 95% CI: 1.168-3.253; p = 0.011), low TIMI grade (≤ 1) before percutaneous coronary intervention (OR: 1.100; 95% CI: 1.086-1.257; p < 0.01), high thrombus load (OR: 1.274; 95% CI: 1.423-2.761; p = 0.030), and long target lesion (OR: 1.948; 95% CI: 1.908-1.990; p = 0.019) were independent risk factors. CONCLUSIONS: No-reflow phenomenon after percutaneous coronary intervention in patients with acute coronary syndrome was affected by complicated pathological factors.

Optimization of beta-carotene production by a newly isolated Serratia marcescens strain

beta-carotene is a commonly used food colorant. In this work, a novel beta-carotene producing strain, Serratia marcescens RB3, was isolated and identified by physiological and biochemical tests, as well as 16S rDNA sequence analysis. The production of beta-carotene by S. marcescens RB3 was identified through HPLC analysis. The cultivation conditions for beta-carotene production by S. marcescens RB3 were optimized as 2.0 percent lactose, 2.0 percent peptone, 0.3 percent beef extract, 1.0 percent NaCl supplemented with 0.05 percent Fe2+, pH 6.0 and 30ºC. Under the optimal conditions, the yield of beta-carotene achieved 2.45 ug/mL.