A novel gene (ANK58566) encoding a cold-active α-amylase was cloned from marine bacterium Bacillus sp. dsh19-1 (CCTCC AB 2015426), and the protein was expressed in Escherichia coli. The gene had a length of 1302 bp and encoded an α-amylase of 433 amino acids with an estimated molecular mass of 50.1 kDa. The recombinant α-amylase (AmyD-1) showed maximum activity at 20 °C and pH 6.0, and retained about 35.7% of activity at 4 °C. The AmyD-1 activity was stimulated by Ca and Na. However, the chelating agent, EDTA, inactivated the enzyme. Moreover, AmyD-1 displayed extreme salt tolerance, with the highest activity in the presence of 2.0 M NaCl and 60.5% of activity in 5.0 M NaCl. The K, V and k of AmyD-1 in 2.0 M NaCl were 2.8 mg ml, 21.8 mg ml min and 933.5 s, respectively, at 20 °C and pH 6.0 with soluble starch as substrate. MALDI-TOF MS (Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry) revealed that the end products of starch hydrolysis by AmyD-1 were glucose, maltose, maltotriose, maltotetraose, and malt oligosaccharides. Thus, AmyD-1 is one of the very few α-amylases that can tolerate low temperatures and high salt concentrations, which makes it to be a potential candidate for research in basic and applied microbiology.
To address the deficient activity of TrCel5A in naturally secreted cellulase preparation, this study used the GAP promoter to induce constitutive expression of Trichoderma reesei TrCel5A in Pichia pastoris. A recombinant TrCel5A was screened out after gene optimization, synthesis, and expression. The biochemical and enzymatic properties of the new recombinant were characterized. As a result, optimization of shake-flask fermentation of the recombinant was obtained at 28 °C, 2% inoculum volume, an initial pH of 6.0, as well as glycerol and Tween-80 additions of 30 g/L and 6 g/L, respectively. Under the above-optimized conditions, the recombinant produced 14.8 U/mL of the enzyme activity at 96 h of fermentation. To further enhance enzyme production, pilot-scale cultivation was evaluated using 5-L bioreactors. Using high-cell-density fermentation, the recombinant strain increased enzyme activity to 130.4 U/ml and protein content to 2.49 g/L. In addition, the kinetic factors, including K m and V max values for TrCel5A, were detected to be 5.1 mg/mL and 265.9 μmol/(min . mg), respectively. Thus, TrCel5A was effectively expressed in P.pastoris under the GAP promoter, and it demonstrated its potential in commercially relevant enzyme hydrolysis of lignocellulosic biomass.
Linolenic acid is an important fatty acid, and volatiles generated by its oxidation are the major components of food flavor. In this study, volatile components generated from linolenic acid during heating were detected and analyzed by using thermal desorption cryo-trapping system coupled with gas chromatography-mass spectrometry. A total of 52 volatile compounds were identified, including aldehydes (18), ketones (12), alcohols (6), furans (4), acids (6) and aromatic compounds (6). The forming temperature of each volatile compound was also determined. It was found that most volatile compounds with shorter carbon chains were mainly generated at lower temperatures, while volatile compounds with longer carbon chains were mainly produced at higher temperatures. Results of principal component analysis show that most of the identified volatiles were considered as the characteristic ones of the high temperature points. Potential thermal oxidation mechanism of linolenic acid was also given at the same time.
Amorphadiene is the precursor to synthesize the antimalarial drug artemisinin. The production of amorphadiene and artemisinin from metabolically engineered microbes may provide an alternate to plant secondary metabolite extraction. Microbial consortia can offer division of labor, and microbial co-culture system can be leveraged to achieve cost-efficient production of natural products. Using a co-culture system of Y. lipolytica Po1f and Po1g strains, subcellular localization of ADS gene (encoding amorphadiene synthase) into the endoplasmic reticulum, co-utilization of mixed carbon source, and enlargement of the endoplasmic reticulum (ER) surface area, we were able to significantly improve amorphadiene production in this work. Using Po1g/PPtM and Po1f/AaADSERx3/iGFMPDU strains and co-utilization of 5 µM sodium acetate with 20 g/L glucose in YPD media, amorphadiene titer were increased to 65.094 mg/L. The enlargement of the ER surface area caused by the deletion of the PAH1 gene provided more subcellular ER space for the action of the ADS-tagged gene. It further increased the amorphadiene production to 71.74 mg/L. The results demonstrated that the importance of the spatial localization of critical enzymes, and manipulating metabolic flux in the co-culture of Y. lipolytica can be efficient over a single culture for the bioproduction of isoprenoid-related secondary metabolites in a modular manner. Graphical Abstract
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