The morphology of Trichoderma reesei is a vitally important factor for cellulase productivity. This study investigated the effect of hyphal morphology on cellulase production in the hyper-cellulolytic mutant, T. reesei DES-15. With a distinct morphology, T. reesei DES-15 was obtained through Diethyl sulfite (DES) mutagenesis. The hyphal morphology of DES-15 batch-cultured in a 5-L fermentor was significantly shorter and more branched than the parental strain RUT C30. The cellulase production of DES-15 during batch fermentation was 66 % greater than that of RUT C30 when cultured the same conditions. DES-15 secreted nearly 50 % more protein than RUT C30. The gene expression level of a set of genes (cla4, spa2, ras2, ras1, rhoA, cdc42, and racA) known to be involved in hyphae growth and hyphal branching was measured by quantitative real-time PCR. The transcriptional analysis of these genes demonstrated that a decrease in gene expressions might contribute to the increased hyphal branching seen in DES-15. These results indicated that the highly branching hyphae in DES-15 resulted in increased cellulase production, suggesting that DES-15 may be a good candidate for use in the large-scale production of cellulase.
Chloroplast formation is associated with embryo development and seedling growth. However, the relationship between chloroplast differentiation and embryo development remains unclear. Five FtsHi genes that encode proteins with high similarity to FtsH proteins, but lack Zn2+-binding motifs, are present in the Arabidopsis genome. In this study, we showed that T-DNA insertion mutations in the Arabidopsis FtsHi4 gene resulted in embryo arrest at the globular-to-heart–shaped transition stage. Transmission electron microscopic analyses revealed abnormal plastid differentiation with a severe defect in thylakoid formation in the mutant embryos. Immunocytological studies demonstrated that FtsHi4 localized in chloroplasts as a thylakoid membrane-associated protein, supporting its essential role in thylakoid membrane formation. We further showed that FtsHi4 forms protein complexes, and that there was a significant reduction in the accumulation of D2 and PsbO (two photosystem II proteins) in mutant ovules. The role of FtsHi4 in chloroplast development was confirmed using an RNA-interfering approach. Additionally, mutations in other FtsHi genes including FtsHi1, FtsHi2, and FtsHi5 caused phenotypic abnormalities similar to ftshi4 with respect to plastid differentiation during embryogenesis. Taken together, our data suggest that FtsHi4, together with FtsHi1, FtsHi2, and FtsHi5 are essential for chloroplast development in Arabidopsis.
Searching for viable strategies to accelerate the catalytic cycle of glycoside hydrolase family 7 (GH7) cellobiohydrolase I (CBHI)—the workhorse cellulose-degrading enzymes, we have performed a total of 12-μs molecular dynamics simulations on GH7 CBHI, which brought to light a new mechanism for cellobiose expulsion, coined "claw-arm" action. The loop flanking the product binding site plays the role of a flexible "arm" extending toward cellobiose, and residue Thr389 of this loop acts as a "claw" that captures cellobiose. Five mutations of residue Thr389 were considered to enhance the loop-cellobiose interaction. The lysine mutant was found to significantly accelerate cellobiose expulsion and facilitate polysaccharide-chain translocation. Lysine mutation of Thr393 in Talaromyces emersonii CBHI (TeCel7A) performed similarly. Lysine approaches the catalytic area and stabilizes the Michaelis complex, potentially affecting glycosylation, the rate-limiting step of the catalytic cycle. QM/MM calculations indicate that lysine replacement diminishes the barrier against proton transfer, the crucial step of glycosylation, by 2.3 kcal/mol. Experimental validation was performed using the full-length wild-type (WT) of TeCel7A and its mutants, recombinantly expressed in Pichia pastoris, to degrade the substrates. Compared with the WT, the lysine mutant revealed an associated higher enzymatic reaction rate. Furthermore, cellobiose yield was also increased by lysine mutation, indicating that dissociation of the enzyme from cellulose was accelerated, which largely stems from the enhanced flexibility of the "arm". The present work is envisioned to help design strategies for improving enzymatic activity, while decreasing enzyme cost.
A UV photodiode fabricated by the UV oxidation of a metallic zinc thin film on p-Si has manifested unique photoresponse characteristics. The electron concentration found by the Hall measurement was 3 × 1016 cm−3, and such a low electron concentration resulted in a low visible photoluminescence. UV illumination enhances the oxidation at low temperatures and decreases the concentration of the oxygen vacancies. The I-V characteristic showed a good rectification with a four-order magnitude difference in the forward and reverse currents at 2 V, and its linear and frequency independent C−2–V characteristic confirmed an abrupt pn junction. The photoresponse showed a visible blindness with a responsivity ratio of UV and visible light as high as 100. Such a visible-blind photoresponse was attributed to the optimum thickness of the SiO2 formed on the Si surface during the UV oxidation at 400 °C. A lower potential barrier to holes at the ZnO/SiO2 interface facilitates Fowler-Nordheim tunneling of the photo-generated holes during the UV illumination, while a higher potential barrier to electrons efficiently blocks transport of the photo-generated electrons to the ZnO during the visible light illumination. The presence of oxide resulted in a slow photoresponse to the turn-on and off of the UV light. A detailed analysis is presented to understand how the photo-generated carriers contribute step by step to the photocurrent. In addition to the slow photoresponse associated with the SiO2 interfacial layer, the decay of the photocurrent was found extremely slow after turn-off of the UV light. Such a slow decay of the photocurrent is referred to as a persistent photoconductivity, which is caused by metastable deep levels. It is hypothesized that Zn vacancies form such a deep level, and that the photo-generated electrons need to overcome a thermal-energy barrier for capture. The ZnO film by the UV oxidation at 400 °C was found to be rich in oxygen and deficient in zinc.
Nickel oxide (NiO) is one of few metal-oxide semiconductors showing a p-type conductivity and can be combined with an n-type semiconductor to fabricate a pn diode. In this study, isotype and anisotype heterojunction diodes were fabricated by ultraviolet (UV) oxidation at 350 °C of metallic Ni deposited on p- and n-Si substrates. The I-V characteristics of the NiO/p-Si diode do not show rectification, while those of the NiO/n-Si diode show rectification with a difference of two orders of magnitude between the currents at 2 and −2 V. The linear C−2 -V characteristics of the NiO/n-Si and secondary ion mass spectroscopy profile confirm that the UV oxidation temperature is low enough so as not to affect deep inside the Si. The NiO layers formed by the oxidation of Ni under UV illumination are found to be more conductive and more suitable for the diode fabrication than those by the thermal oxidation of Ni without UV illumination at the same temperature. Because the oxidation temperature is relatively low and can be further reduced, the UV oxidation can become an important process technology to form various metal-oxide semiconductors from printed metals for future printed flexible electronics.
'/%3e%3cuse xlink:href='%23a' transform='rotate(23.036 .093 25.536)'/%3e%3cuse xlink:href='%23a' transform='rotate(45.87 1.273 16.18)'/%3e%3cuse xlink:href='%23a' transform='rotate(69.945 .996 12.078)'/%3e%3cuse xlink:href='%23a' transform='rotate(20.66 -19.689 31.932)'/%3e%3c/svg%3e)