Loss of second and sixth conserved cysteine residues from trypsin inhibitor-like cysteine-rich domain-type protease inhibitors in Bombyx mori may induce activity against microbial proteases

Previous studies have indicated that most trypsin inhibitor-like cysteine-rich domain (TIL)-type protease inhibitors, which contain a single TIL domain with ten conserved cysteines, inhibit cathepsin, trypsin, chymotrypsin, or elastase. Our recent findings suggest that Cys^2nd and Cys^6th were lost from the TIL domain of the fungal-resistance factors in Bombyx mori, BmSPI38 and BmSPI39, which inhibit microbial proteases and the germination of Beauveria bassiana conidia. To reveal the significance of these two missing cysteines in relation to the structure and function of TIL-type protease inhibitors in B. mori, cysteines were introduced at these two positions (D36 and L56 in BmSPI38, D38 and L58 in BmSPI39) by site-directed mutagenesis. The homology structure model of TIL domain of the wild-type and mutated form of BmSPI39 showed that two cysteine mutations may cause incorrect disulfide bond formation of B. mori TIL-type protease inhibitors. The results of Far-UV circular dichroism (CD) spectra indicated that both the wild-type and mutated form of BmSPI39 harbored predominantly random coil structures, and had slightly different secondary structure compositions. SDS-PAGE and Western blotting analysis showed that cysteine mutations affected the multimerization states and electrophoretic mobility of BmSPI38 and BmSPI39. Activity staining and protease inhibition assays showed that the introduction of cysteine mutations dramaticly reduced the activity of inhibitors against microbial proteases, such as subtilisin A from Bacillus licheniformis, protease K from Engyodontium album, protease from Aspergillus melleus. We also systematically analyzed the key residue sites, which may greatly influence the specificity and potency of TIL-type protease inhibitors. We found that the two missing cysteines in B. mori TIL-type protease inhibitors might be crucial for their inhibitory activities against microbial proteases. The genetic engineering of TIL-type protease inhibitors may be applied in both health care and agricultural industries, and could lead to new methods for breeding fungus-resistant transgenic crops and antifungal transgenic silkworm strains.     

Identification of soil quality indicators for assessing the effect of different tillage practices through a soil quality index in a semi-arid environment

Tillage is known to potentially affect soil quality in various ways. In this study, a soil quality index (SQI) was developed by quantifying several soil attributes either sensitive or insensitive to physical disturbance, using factor analysis as a dimension reduction technique, in order to discriminate different tillage systems. Soil properties including physical (MWD), chemical (pH, organic C, total N, available P and POM contents) and microbial (MBC, MBN, PCM, PNM and three enzymes) parameters were measured to establish a minimum data set (MDS) for the assessment of overall SQI. The soil attributes were determined on samples (0–20 cm depth) collected under moldboard (MP) and disk (DP) plows as conventional tillage (CT), and rotary (RP) and chisel (CP) plows as reduced tillage (RT) systems with a similar plant C input rate and cover crop over a period of six years (2005–2011) in a semi-arid calcareous soil (Calcixerepts) from Central Iran. Results indicated a clear difference in soil quality among the tillage systems with a significant increase of SQI under RT over time, particularly under CP practices. Although RT improved most soil microbial attributes, not all attributes contributed to SQI because of their close interrelationship. The final SQI consisted only of geometric mean of microbial activity (GMA, the square root of the product of PCM and PNM) and geometric mean of enzyme activity (GME, the cube root of the product of enzyme activities). Soil GME and GMA were found to be as key indicators contributing 55% and 36% to SQI, respectively. Therefore, the GME and GMA were the most important indicators effectively discriminating tillage systems, and could be used to monitor the enhancement of soil quality under RT in this semiarid environment. The influence of tillage year on SQI was greater than that of tillage practices. In conclusion, RT systems were characterized by a higher value of SQI, suggesting a good recovery of soil capacity and functions after abandoning CT in the studied area. Smallholder farmers should therefore be aware of the potential for high soil quality in future as a result of continuing RT systems, especially with surface tillage using CP practices.     

Operation of a Benchtop Bioreactor

Fermentation systems are used to provide an optimal growth environment for many different types of cell cultures. The ability afforded by fermentors to carefully control temperature, pH, and dissolved oxygen concentrations in particular makes them essential to efficient large scale growth and expression of fermentation products. This video will briefly describe the advantages of the fermentor over the shake flask. It will also identify key components of a typical benchtop fermentation system and give basic instruction on setup of the vessel and calibration of its probes. The viewer will be familiarized with the sterilization process and shown how to inoculate the growth medium in the vessel with culture. Basic concepts of operation, sampling, and harvesting will also be demonstrated. Simple data analysis and system cleanup will also be discussed.     

N-Linked Oligosaccharides of Aspergillus awamori Feruloyl Esterase Are Important for Thermostability and Catalysis

A unique N-linked glycosylation motif (Asn⁷⁹-Tyr-Thr) was found in the sequence of type-A feruloyl esterases from Aspergillus spp. To clarify the function of the flap, the role of N-linked oligosaccharides located in the flap region on the biochemical properties of feruloyl esterase (AwFAEA) from Aspergillus awamori expressed in Pichia pastoris was analyzed by removing the N-linked glycosylation recognition site by site-directed mutagenesis. N79 was replaced with A or Q. N-glycosylation-free N79A and N79Q mutant enzymes had lower activity than that of the glycosylated recombinant AwFAEA wild-type enzyme toward α-naphthylbutyrate (C4), α-naphthylcaprylate (C8), and phenolic acid methyl esters. Kinetic analysis of the mutant enzymes indicated that the lower catalytic efficiency was due to a combination of increased K _m and decreased k _cat for N79A, and to a considerably decreased k _cat for N79Q. N79A and N79Q mutant enzymes also exhibited considerably reduced thermostability relative to the wild-type.     

Genetic characterisation of a further homoeoallelic series of grain esterase loci,Est-6, in wheat

Summary Isoelectric focussing in alkaline pH gels has permitted the identification of a new homoeoallelic series of genes,Est-6, encoding grain esterases in bread wheat,Triticum aestivum. Nullisomic analysis located these genes to the short arms of the homoeologous group 2 chromosomes. A search for polymorphism withinEst-6 revealed null alleles at each ofEst-A6,Est-B6 andEst-D6. A further homoeolocus,Est-M6, is present on chromosome arm2MS ofAegilops comosa.     

Enantioselective hydrolysis of esters of secondary alcohols using lyophilized Bakers'' yeast

A number of acetoxycarboxylic acid esters were hydrolysed enantioselectively by Saccharomyces cerevisiae Hansen leading to chiral hydroxycarboxylic acid esters of high optical purity. The scope and limitations of this method with respect to the substitutional pattern of substrates were investigated. Subcellular localization of the hydrolytic activity on the plasma membrane led to the assumption that unspecific carboxyl esterases are responsible for hydrolysis of this type of substrate. Comparative experiments using viable cells and lyophilized cells as source of enzyme revealed the latter to be superior with respect to enantioselection and ease of handling.     

Secretion of Mucor rennin, a fungal aspartic protease of Mucor pusillus, by recombinant yeast cells

Summary The aspartic protease gene of a zygomycete fungus Mucor pusillus was expressed in Saccharomyces cerevisiae under the control of the yeast GAL7 promoter. A putative preproenzyme with an NH₂-terminal extension of 66 amino acids directed by the gene was processed in yeast cells and the mature enzyme, whose NH₂-terminus was identical to that of the Mucor enzyme, was efficiently secreted into the medium at a concentration exceeding 150 mg/l. The enzyme secreted from the recombinant yeast was more glycosylated than the native Mucor enzyme but its enzymatic properties were almost identical with those of the native enzyme, which has been used as a milk coagulant in cheese manufacture.