摘要：
Production of selenium nanoparticles (SeNPs) by chemical approaches is costly and environmentally hazardous, whereas biological methods, especially those using bacteria, can avoid these disadvantages. In this study, rapid biogenic synthesis of SeNPs by a heavy metal-resistant bacterium Stenotrophomonas sp., strain EGS12 was investigated. SeNPs were characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), and energy-dispersive X-ray spectroscopy (EDX). TEM and SEM analysis showed that the SeNPs were spherical and ranged from 100 to 200 nm in size. EDX demonstrated that the SeNPs contained C, Se, O, P, and S, and the content of Se was 32.64%. The mechanism of SeNPs biogenic synthesis by this strain was also investigated. The results documented that the biogenic synthesis of SeNPs, consisting of reduction of selenite to elemental selenium, was conducted synergistically by two reductases: nitrite reductase and thiol-mediated reductase. Thus, a novel pathway of efficient SeNP biosynthesis by a heavy metal-resistant Stenotrophomonas genus strain was identified.
关键词：
mannanase;rational design;Brevibacillus brevis;cell-free protein synthesis
摘要：
BACKGROUNDMannanases are important enzymes which are widely used as a tool in agriculture and food industries. To improve the performance of mannanase Man23, a mutant library was created with rational design, and mutations were introduced on loops around the catalytic region. The Brevibacillus brevisB16 cell-free system which was created in this experiment provided the ability to express the mutant library efficiently. The activities of mutants were measured with a multi-volume spectrophotometer. RESULTSThe mutant Man1606 gained from this system is a sextet which has mutations of N146G, S147H, S156P, T157Y, Q206S and T249H simultaneously on loops 6, 8 and 10. Man1606 showed higher activity and stability than Man23. The optimal temperature of Man1606 rose by 5 degrees C (from 55 to 60 degrees C) and the optimal pH increased slightly but its range became broader. CONCLUSIONThis experiment demonstrated the B. brevis cell-free system shortens the expression time and is an efficient tool for mannanase engineering. (c) 2016 Society of Chemical Industry
摘要：
BACKGROUNDThe disordered residues on distal loops affect the molecular structural stability and on some occasions have regulatory roles in catalytic reaction. To increase understanding of the influence of distal residue mutation, this study explored the thermostability and enzymatic activity of mannanase Man1312 deletion mutants. The focus was on residues located on the N-terminal region because they are more disordered and changeable. The effects of N-terminal truncation on enzymatic activity and thermal dynamics were investigated by spectrophotometry, circular dichroism and differential scanning calorimetry assays. RESULTSThe deletion mutants on V3, N7 and Q11 showed a marked increase in stability, while the enzymatic activity was significantly improved when triplet deletion was carried out. Triplet deletion MandVNQ showed around double the stability of its corresponding single-site and double-site deletion mutants. The T-m value of MandVNP was about 8 degrees C higher than that of Man1312. MandVNP had improved characteristics of T-opt by 10 degrees C, t(1/2) by 10min and catalytic activity by 11% in comparison with Man1312. Analysis of spectra and modeling showed that MandVNQ had increased helix and strand contents. CONCLUSIONN-terminal truncation had positive effects on the thermostability and activity of mannanase. (c) 2015 Society of Chemical Industry
关键词：
Mannanase;Recombinant gene;Host bacterium;Expression system
摘要：
Mannanase is an enzyme that can catalyze random hydrolysis of beta-1,4-mannosidic linkages in the main chain of mannans, glucomannans and galactomannans which are the key polymers in hemicellulose. It has been used in a number of different industrial applications including food, feed, pharmaceutical, pulp/paper industries, and second generation biofuel. To optimize the expression system of mannanase Man23 gene, two kinds of vectors and host bacteria were determined and compared. Recombinants pHY-p43-man23 and pBPS-man23 were constructed and transferred into Bacillus subtilis WB600 and Brevibacillus brevis respectively. For mannanase Man23 gene, recombinant pHY-p43-man23 expressed in Brevibacillus brevis had higher production and activity. Compared to the wild-type Bacillus subtilis B23, the production of recombinant pHY-p43-man23 in B. brevis increased by 10 times and activity increased by 21.3%. pHY-p43-man23 in B. brevis had activity at the range of 20 ~ 70°C but its optimum temperature was 50°C and had activity from pH 4 ~ 10 but its optimum pH was around 7. This demonstrated the recombinant had improved stability as well. Mannanase is an important industrial enzyme and combination of vector pHY-p43 and host Brevibacillus brevis is a novel expression system for a mannanase decoding gene. This work aims at exploring a better expression system of mannanase Man23 decoding gene for industrial application.
