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Cephalosporin C (CPC) acylase is important for the one-step production of 7-aminocephalosporanic acid (7-ACA), a key intermediate for cephalosporin antibiotics. However, its application is hampered by the low activity, substrate inhibition, and product inhibition. In this study, two rounds of combinatorial active-site saturation testing (CASTing) were carried out on the CPC acylase acyII from Pseudomonas SE83, and one mutant H57βA/H70βY with no substrate inhibition was obtained. For further engineering to reduce the product inhibition, a quick pH indicator assay was developed, allowing for real-time monitoring of the product inhibition in the presence of added 7-ACA. The utility of the assay was demonstrated by screening six libraries of site-directed saturation mutagenesis libraries of H57βA/H70βY. A new mutant H57βA/H70βY/I176βN was obtained, which showed a k 3.26-fold and a K 3.08-fold that of the wild type, respectively. Given the commercial value of the enzyme, both this pH indicator assay and the triple mutant should be useful for further engineering of the enzyme to increase the specific activity and to decrease the product inhibition. [ABSTRACT FROM AUTHOR]

Abstract: A new hot rolled titanium-microalloyed steel with yield strength of 700MPa has been developed by CSP (compact strip production) process based on commercial weather resistant steel. EBSD results showed that the average size of its grains with high angle boundaries (>15°) was 3.3μm. High-density dislocations and large number of nanometer particles were observed in the steel product by TEM. X-ray analysis on the electrolytically extracted phase from the steel indicated that fraction of MX phase was 0.0793wt%, in which the particles smaller than 10nm accounted for 33.7%. The contribution of precipitation hardening resulting from nanometer particles was calculated as approximate 158MPa. The commercial weather resistant steel, reference steel for comparison with 450MPa yield strength, was also prepared and investigated. It can be concluded that grain refinement is still a major strengthening mechanism in this high strength steel, but precipitation hardening of nanometer TiC precipitates is the dominant factor to increasing the yield strength in new developed steel compared with the reference steel. [Copyright &y& Elsevier]

Abstract: Bovine follicle-stimulating hormone (bFSH) is a pituitary gonadotropin composed of two non-covalently associated polypeptide subunits, which must be glycosylated, folded, and assembled as a heterodimer to be biologically active. Low-level expression of the recombinant bFSH is the factor that limits its usefulness as a superovulation treatment for cows. To increase the production of recombinant bFSH, human protein disulfide isomerase (hPDI) was expressed simultaneously in engineered Pichia strains. The secretion characteristics of bFSH with or without hPDI were examined. The co-expression of bFSH and hPDI is increased to 1.56mg/l of heterodimer in the culture medium, which is 6-fold higher when compared with the control strain carrying the bFSH gene only. These results may be generally applicable to increase the expression of other glycoprotein hormones in yeast. [Copyright &y& Elsevier]

The purpose of this study was to examine the variations in the microbial community structure of soil actinomycetes in fields with continuous cropping of cotton in Xinjiang Autonomous Region, China. Soil samples were collected from four depths in fields with 7-year continuous cotton cropping. The community structure of soil actinomycetes was examined using the 16S rRNA-based polymerase chain reaction-density gradient gel electrophoresis (PCR-DGGE) techniques. The microbial diversity indices of the soil samples from different depths generally decreased along with the period of continuous cotton cropping. When the period of continuous cropping of cotton reached 5 years, the diversity indices rose again and gradually stabilized at a level slightly lower than that of soils with original ecology (i.e., 0-year cotton cropping). Cluster analysis showed that at the 1-20-cm depth, the actinomycete community structure of the soil subjected to 1-year cotton cropping was similar to that of soil subjected to 0-year cotton cropping, whereas that of soils after 3-year continuous cotton cropping showed high similarity. At the 21-40-cm depth, the actinomycete community structure showed various changes but generally recovered to its original pattern after repeated fluctuations. Principal component analysis showed that at the 1-30-cm depth, the actinomycete community structure varied similarly regardless of the period of continuous cotton cropping. In contrast, there were no clear actinomycete community structure variation trends at the 31-40-cm soil depth. Homology comparison of sequences recovered from the DGGE bands showed that the obtained sequences shared similarities >88 %. Alignment with the known homologous sequences indicated a lack of microorganisms related to soil-borne cotton diseases. Continuous cotton cropping exerted significant influences on the community structure of soil actinomycetes in Xinjiang Autonomous Region, which were largely determined by the soil depth and the period of continuous cotton cropping. The microbial diversity of soil actinomycete communities gradually recovered after 5-year continuous cropping. Thereafter, a new actinomycete community structure that was beneficial for continuous cropping of cotton was formed and stabilized each year. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the fungal diversity and its temporal and spatial dynamics in the rhizosphere soil of healthy cotton by high-throughput sequencing. We studied species richness, composition, and distribution of cotton rhizosphere fungal community with respect to location (Alaer, Kuerle, Tumushuke, Hami, Shihezi, Wusu, and Jinghe) and plant growth period (seedling stage, bud stage, flowering stage, and boll-opening stage) using the methods of PCR-based high-throughput sequencing and real-time quantitative PCR. A total of 1,838,454 fungal nuclear ribosomal internal transcribed spacer region sequences (rRNA ITS) were obtained from all cotton plants sampled at different growth stages in the seven locations in Xinjiang. The most abundant fungal group in the cotton rhizosphere was the Ascomycota (78.72%), followed by the Zygomycota (9.56%) and Basidiomycota (2.77%). These sequences revealed an enormous number of operational taxonomic units (OTUs) in cotton (1802 unique OTUs), with 67–464 OTUs in a single cotton sample, at a 3% threshold and a sequencing depth of 30,000 sequences. We identified 33 classes and 389 genera from the resulting 1,800,714 sequences. Sordariomycetes was the most frequent class in all samples, followed by Leotiomycetes and Eurotiomycetes. There were some differences in OTUs among different growth stages, but the differences were not significant, with 382 OTUs (14.66%) being common to each of the stages. A marked difference in the diversity of fungi in the rhizosphere soil of cotton was evident among the different locations, with the highest number of OTUs being detected in Jinghe (1084 OTUs) and clusters of OTUs representative of northern and eastern Xinjiang being detected. There were significantly more tags of Mortierella in Jinghe and Wusu than in the other sampling sites. The dynamics of the rhizosphere fungal communities were influenced by sampling sites. To the best of our knowledge, the current study is the first application of PCR-based Illumina to characterize and compare the fungal biodiversity in multiple rhizosphere soil samples from cotton. [ABSTRACT FROM AUTHOR]

Endogenous bacteria are important for maintaining the health and other ecologically relevant functions of cotton plants. However, little is known about the community structures and diversity of endophytic bacteria in cotton plants. In our study, we used the Illumina amplicon sequencing technology to study the endophytic bacteria found in cotton root tissue in Xinjiang, China. A total of 60.84 × 106 effective sequences of the 16S rRNA gene in the V5–V6 variable region revealed a large number of operational taxonomic units (OTUs), namely 81–338 OTUs, at a cut-off level of 3% and a sequencing depth of 50 000 sequences. Among the 23 classes identified, Gammaproteobacteria was the dominant group, followed by Alphaproteobacteria, Actinobacteria, and Bacillus. The diversity of endogenous bacteria differed at different growth periods, with the most OTUs detected in seedlings (654), followed by the budding stage (381), flowering stage (350), and flocking stage (351). A total of 217 OTUs were common to all four stages. Pantoea tags were more common to the Shihezi region, whereas Erwinia labels were more common to the Hami region. These results suggest that the dynamics of endophytic bacterial communities are affected by plant growth stage. This highlights the relevance of microbial diversity studies in improving our understanding of endophyte communities. [ABSTRACT FROM AUTHOR]

Rhizosphere bacteria are key determinants of plant health and productivity. In this study, we used PCR-based next-generation sequencing to reveal the diversity and community composition of bacteria in the cotton rhizosphere from samples collected in Xinjiang Province, China. We identified 125 bacterial classes within 49 phyla from these samples. Proteobacteria (33.07% of total sequences), Acidobacteria (19.88%), and Gemmatimonadetes (11.19%) dominated the bacterial community. Marked differences were evident in the α-diversity of rhizosphere bacteria during different cotton plant growth and development stages. The operational taxonomic unit (OTU) numbers were highest in seedling and bud stages and decreased at the flowering and fruit-boll-opening stages. Forty-three OTUs from the Proteobacteria were common to all four periods of cotton development. Proteobacteria were more abundant in the rhizospheres of cotton from southern Xinjiang than from northern Xinjiang, while the opposite trend was observed for Acidobacteria. Gemmatimonadetes frequency was broadly the same in both northern and southern Xinjiang. These results suggest that there is abundant diversity in the microbiota of cotton rhizosphere soil. Proteobacteria and Actinobacteria dominated this microbial niche and bacterial communities in the seedling, bud, flowering, and boll-opening stages appear to be more similar to one another than to communities at the other growth stages. [ABSTRACT FROM AUTHOR]

We studied species richness, composition and distribution of sugar beet endophytic fungi with location, plant species and plant growth period using the methods of PCR-based 454 pyrosequencing and terminal-restriction fragment length polymorphism. In total 4345 fungal nuclear ribosomal internal transcribed spacer region sequences (rDNA ITS) obtained from all sugar beet grown in two locations (Shihezi and Changji) on the north slope of Tianshan Mountain, China. The most abundant fungal groups in all sugar beet were Basidiomycota (97.26%). The other dominant phyla were from Ascomycota (0.61%) and Glomeromycota (2.13%). There was a marked difference in diversity of endophytic fungi in sugar beet for different growth stages. The greatest number of Operational T-RFLP Units was detected during sucrose accumulation (84) and tuber growth (74). Endophytic fungal diversity was reduced during seedling growth (18 OTUs) and rosette formation (58 OTUs). One OTU was common to all four periods of growth. There were more OTUs in Shihezi than in Changji. The dynamics of endophytic fungal communities were influenced by plant genotype and plant growth stage. Results indicated that the three factors were significantly related (α = 0.05) to the distribution of endophytic fungi, with host species the most important, followed by sampling dates and sampling locations. To our knowledge, this study is the first application of 454 pyrosequencing and T-RFLP to characterize and compare multiple sugar beet samples. [ABSTRACT FROM AUTHOR]

A new method for isothermal precipitation kinetics of carbides in undercooled austenite and ferrite has been established based on measuring the strength increments. Precipitation-time-temperature (PTT) diagrams of carbides were determined with this method performed on a Gleeble simulator, for a low-carbon titanium microalloyed steel in a temperature range from 530 °C to 670 °C. Fine precipitates were studied by using high resolution transmission electron microscope (HRTEM), and their volume fractions together with their attributions to strength increments were calculated and analyzed. Results show that the strength increments are mainly attributed to the γ/α interphase precipitation whose nano-scale TiC precipitates obey the Baker-Nutting orientation relationship with respect to the ferrite matrix: (100) TiC ǁ (100) α-Fe and [011] TiC ǁ [001] α-Fe . The PTT curves obtained have a classic C-shape with their noses at about 600 °C, where the strength increment reaches 114 MPa after isothermally holding for 1 h. Additionally, the corresponding precipitation kinetics curves are presented in an S-shape, and a modified-form of Avrami formula is introduced to describe their growth periods. The presented mathematical expression fits the experimental data quite well. Consequently, the proposed new method is intuitive, effective and convenient, particularly suitable for detecting the precipitation kinetics in low temperature range. [ABSTRACT FROM AUTHOR]

Titanium microalloyed low carbon steels were rolled into plates by controlled rolling process, followed by two subsequent cooling routes, including air cooling (AC) directly and isothermal treatment (IT) at 600 °C for 1 h. Fine precipitates were studied by high-resolution transmission electron microscope with energy dispersive X-ray spectroscope, as well as X-ray diffraction. It is noted that a larger amount of nano-sized TiC precipitated in the matrix of IT steel, and their orientation relationships with respect to the ferrite matrix were ( 100 ) T i C ∥ ( 110 ) α − F e and [ 011 ] T i C ∥ [ 1 ¯ 11 ] α − F e for strain-induced precipitated TiC of 10–40 nm in size, and ( 100 ) T i C ∥ ( 100 ) α − F e and [ 011 ] T i C ∥ [ 001 ] α − F e for random or interphase precipitated TiC of 1–10 nm in size. Contributions to the yield strength associated with grain refinement, solid solution, dislocation and precipitation were analyzed by using a modified form of Hall–Petch, Taylor, and Ashby–Orowan relationship, respectively. It is revealed that isothermal treatment is much advantageous to precipitation and the major increase in yield stress is mainly attributed to the precipitation strengthening of nano-sized TiC. [ABSTRACT FROM AUTHOR]