Biodegradation of Lignocellulose by White-Rot Fungi: Structural Characterization of Water-Soluble Hemicelluloses

In the biological pretreatment process, white-rot fungi are mostly used to degrade lignin and carbohydrates in lignocellulosic biomass. In this study, water-soluble hemicelluloses were recovered from birch wood (Betula alnoides) decayed by white-rot fungi (Ganoderma lucidum C7016) for different durations up to 16 weeks. Accordingly, the dimethyl sulfoxide (DMSO)-soluble hemicelluloses were isolated from the untreated birch wood as a comparison. Results showed that the fungal-degraded polysaccharides were acidic hemicelluloses having a high content of uronic acids ranging from 20.6 to 22.5 %. Gel permeation chromatography analysis demonstrated that the recovered water-soluble hemicelluloses had a lower average molecular weight (M _w, 15,990–27,560 g?mol^?1) than that of the DMSO-soluble hemicelluloses (M _w , 33,960 g?mol^?1). Fourier transform infrared spectroscopy, scanning electron microscopy, one- and two-dimensional nuclear magnetic resonance spectroscopy also revealed significantly changes between those of fungal degraded and DMSO-soluble hemicelluloses. It was proposed that the hemicelluloses with low molecular weights were easily removed from wood by fungal degradation. This research revealed the changes of hemicelluloses in fungal degradation in the natural environment, which may enable the exploration of novel methods in bioconversion of lignocellulosic biomass for the production of biofuels and biopolymers, in addition to the development of new and better ways to protect wood from biodegradation by microorganisms.     

饲料中添加溶菌酶对吉富罗非鱼生长、免疫-抗氧化功能及血清抗菌性能的影响

为研究在饲料中添加不同水平的溶菌酶制品对吉富罗非鱼(GIFT, Oreochromis niloticus)生长性能、免疫-抗氧化功能和血清抗菌性能的影响, 选用平均体重为(11.350.08)g的吉富罗非鱼960尾, 随机分为6组(每组4个重复, 每个重复40尾), 分别投喂基础饲料(对照组)和5种添加水平分别为18、36、54、72和90 mg/kg溶菌酶制品的试验饲料, 养殖周期为60d。结果表明: (1) 54 mg/kg溶菌酶添加组鱼的生长性能和饲料利用情况最优, 增重率和蛋白质效率均显著高于对照组, 饲料系数显著低于对照组(P0.05); 肝体比随溶菌酶添加水平的增加呈现下降趋势, 90 mg/kg添加组显著低于对照组(P0.05); 脾脏指数在36和54mg/kg添加组显著低于对照组(P0.05); 全鱼粗蛋白和粗灰分含量在54 mg/kg添加组均呈现较高水平, 显著高于对照组(P0.05)。(2)溶菌酶添加水平对罗非鱼的免疫-抗氧化能力产生影响, 54和72 mg/kg添加水平能显著提高鱼体血清和肝脏的超氧化物歧化酶、过氧化氢酶活性, 降低丙二醛含量(P0.05); 肝脏溶菌酶活性在54和72 mg/kg添加组均显著高于对照组(P0.05), 而血清溶菌酶活性随溶菌酶添加水平的增加呈现下降趋势(L90组除外), 显著低于对照组(P0.05)。(3)血清抗菌试验显示, 54和72 mg/kg溶菌酶添加组罗非鱼对大肠杆菌、金黄色葡萄球菌、嗜水气单胞菌和溶藻弧菌的抑制能力显著高于对照组(P0.05), 而对枯草芽孢杆菌的抵抗能力最低, 比对照组分别低34.71%和42.21% (P0.05)。结果表明, 在本试验条件下, 在吉富罗非鱼饲料中添加54 mg/kg溶菌酶制品可以改善其生长性能; 当添加水平为54和72 mg/kg时, 罗非鱼的免疫-抗氧化能力和血清抗菌性能均得到了显著提高。     

Cloning and over-expression of an alkaline protease from Bacillus licheniformis

The alkaline protease gene, apr, from Bacillus licheniformis 2709 was cloned into a Bacillus shuttle expression vector, pHL, to yield the recombinant plasmid pHL-apr. The pHL-apr was expressed in Bacillus subtilis WB600, yielding a high expression strain BW-016. The amount of alkaline protease produced in the recombinant increased by 65% relative to the original strain. SDS-PAGE analysis indicated a Mr of 30.5 kDa. The amino acid sequence deduced from the DNA sequence analysis revealed a 98% identity to that of Bacillus licheniformis 6816.     

Unassisted refolding of urea-denatured arginine kinase from shrimp Feneropenaeus chinensis: evidence for two equilibrium intermediates in the refolding pathway

The refolding process and the equilibrium intermediates of urea-denatured arginine kinase (AK) were investigated by 1-anilino-8-naphthalenesulfonate (ANS) intrinsic fluorescence, far-UV circular dichroism (CD), size-exclusion chromatography (SEC), and enzymatic activity. In dilute denaturant, two equilibrium refolding intermediates (I and N') were discovered, and a refolding scheme of urea-denatured AK was proposed. During the refolding of urea-denatured AK, the fluorescence intensity increased remarkably, accompanied by a significant blue shift of the emission maximum and a pronounced increase in molar ellipticity of CD at 222 nm. The first folding intermediate (I) was inactive in urea solution ranging between 2.4 and 3.0 M. The second (N') existed between a 0.4- and 0.8-M urea solution, with slightly increased activity. Neither the blue shift emission maximum nor the molar ellipticity of CD at 222 nm showed significant changes in these two regions. The two intermediates were characterized by monitoring the ANS binding ability in various residual urea solutions, and two peaks of the emission intensity were observed in urea solutions of 0.6 and 2.8 M, respectively. The SEC results indicated that a distribution coefficient (K(D)) platform existed in urea solutions ranging between 2.4 and 3.0 M urea, suggesting that there was a similarly apparent protein profile and size in the urea solution region. The refolding kinetics showed that the urea-denatured AK was in two-phase refolding. Proline isomerization occurred in the unfolding process of AK, which blocked the slow phase of refolding. These results suggested that the refolding process of urea-denatured AK contained at the least two equilibrium refolding intermediates.     

Optimization of riboflavin production by recombinant <Emphasis Type="Italic">Bacillus subtilis</Emphasis> RH44 using statistical designs

A sequential optimization strategy, based on statistical experimental designs, was used to enhance the production of riboflavin by recombinant Bacillus subtilis RH44. In the first instance, the medium components were optimized in shake flask cultures. After preliminary experiments of nitrogen source selection, the two-level Plackett–Burman (PB) design was implemented to screen medium components that significantly influence riboflavin production. Among the 15 variables tested, glucose, NaNO₃, K₂HPO₄, ZnSO₄, and MnCl₂ were identified as the most significant factors (confidence levels above 95%) for riboflavin production. The optimal values of these five variables were determined by response surface methodology (RSM) based on the central composite design (CCD). The validity of the model developed was verified, and the optimum medium led to a maximum riboflavin concentration of 6.65 g/l, which was 44.3 and 76.4% higher than the improved medium and the basal medium, respectively. A glucose-limited fed-batch culture profile in a 5-l fermentor was consequently designed according to the above optimum medium in shake flasks. A final riboflavin concentration of 16.36 g/l was obtained in 48 h, which further verified the practicability of this optimum strategy.     

A new strategy for assessing selenoprotein function: siRNA knockdown/knock-in targeting the 3'-UTR

Selenocysteine insertion into protein in mammalian cells requires RNA elements in the 3'-untranslated regions (3'-UTRs) of selenoprotein genes. The occurrence of these conserved sequences should make selenoproteins particularly amenable for knockdown/knock-in strategies to examine selenoprotein functions. Herein, we utilized the 3'-UTR of various selenoproteins to knock down their expression using siRNAs and then knock in expression using constructs containing mutations within the target region. Thioredoxin reductase 1 (TR1) knockdown in a mouse kidney cell line resulted in the cells growing about 10% more slowly, being more sensitive to UV radiation, and having increased apoptosis in response to UV than control cells. The knockdown cells transfected with a construct encoding the wild-type TR1 gene and having mutations in the sequences targeted by siRNA restored TR1 expression and catalytic activity, rendered the knockdown cells less sensitive to UV, and protected the cells against apoptosis. We also applied this technique to other selenoproteins, selenophosphate synthetase 2 and glutathione peroxidase 1, and found that mRNA and protein levels were restored following transfection of knockdown cells with the corresponding knock-in constructs. In addition to important new insights into the functions of key mammalian selenoproteins, the data suggest that the RNAi-based knock-in technology could distinguish phenotypes due to off-targeting and provide a new method for examining many of the subtleties of selenoprotein function not available using RNAi technology alone.     

丛枝菌根真菌对滨梅扦插苗生根、生长和抗病相关酶活性的影响

研究Glomus mosseae,G.diaphanum和G.etunicatum对滨梅插条生根、生长和抗病相关酶活性的影响.结果表明:G.mosseae侵染导致最高生根百分率(47.6%),最多次生细根(20.4条),最高的根干重(0.26 g),最高的地上部分干重(3.55 g),最高的幼苗高度(51.3 cm)及...     

Evidence for direct roles of two additional factors, SECp43 and soluble liver antigen, in the selenoprotein synthesis machinery

Selenocysteine (Sec) is inserted into selenoproteins co-translationally with the help of various cis- and trans-acting factors. The specific mechanisms of Sec biosynthesis and insertion into protein in eukaryotic cells, however, are not known. Two proteins, SECp43 and the soluble liver antigen (SLA), were previously reported to interact with tRNA([Ser]Sec), but their functions remained elusive. Herein, we report that knockdown of SECp43 in NIH3T3 or TCMK-1 cells using RNA interference technology resulted in a reduction in the level of methylation at the 2'-hydroxylribosyl moiety in the wobble position (Um34) of Sec tRNA([Ser]Sec), and consequently reduced glutathione peroxidase 1 expression. Double knockdown of SECp43 and SLA resulted in decreased selenoprotein expression. SECp43 formed a complex with Sec tRNA([Ser]Sec) and SLA, and the targeted removal of one of these proteins affected the binding of the other to Sec tRNA([Ser]Sec). SECp43 was located primarily in the nucleus, whereas SLA was found in the cytoplasm. Co-transfection of both proteins resulted in the nuclear translocation of SLA suggesting that SECp43 may also promote shuttling of SLA and Sec tRNA([Ser]Sec) between different cellular compartments. Taken together, these data establish the role of SECp43 and SLA in selenoprotein biosynthesis through interaction with tRNA([Ser]Sec) in a multiprotein complex. The data also reveal a role of SECp43 in regulation of selenoprotein expression by affecting the synthesis of Um34 on tRNA([Ser]Sec) and the intracellular location of SLA.