Professor Christopher Julian Hewitt FREng 1969–2019

Characterization of a glucose tolerant β-glucosidase from Aspergillus unguis with high potential as a blend - in for biomass hydrolyzing enzyme cocktails Abstract Objectives Characterization of glucose tolerant beta glucosidase (GT-BGL) secreted by Aspergillus unguis NII 08123, determination of the gene and protein sequences of the enzyme and establishing its performance in blends for lignocellulose hydrolysis. Results Supplementation of A. unguis beta glucosidase (BGL) to cellulase released 1.6 times more sugar within 12 h during the hydrolysis of lignocellulosic biomass. The enzyme was determined to be similar to BGL-F from Emericella nidulans by MALDI-TOF analysis, and was found to be a GH3 family protein. Molecular Docking simulation studies showed that the enzyme has lesser affinity for glucose (− 5.7 kcal/mol) compared to its substrate cellobiose (− 7.5 kcal/mol). The residues present in the N-terminal domain are mostly involved in bond formation with both the substrate and the product, while the C-terminal domain contains the catalytic region. In-silico studies showed that its predicted structure is unlike that of previously reported BGLs, which might provide a clue to its exceptional catalytic activity. Conclusion The GT-BGL from A. unguis NII 08123 was proven effective as a blend in for biomass hydrolyzing enzyme cocktails and the possible reasons for its glucose tolerance was determined through studies on its modeled structure.

Overexpression of acetyl-CoA carboxylase increases fatty acid production in the green alga Chlamydomonas reinhardtii Abstract Chlamydomonas reinhardtii is a photosynthetic unicellular model algae with multiple biotechnological advantages, and its fatty acids can be used to produce biofuels. Numerous studies suggest that acetyl-coA carboxylase (ACCa) catalyzes the first committed and rate-limiting step of fatty acid biosynthesis, thereby playing a central role in oil accumulation. Here, we cloned and overexpressed ACCa in C. reinhardtii to directly evaluate its effect on fatty acid synthesis. GC–MS analysis found that the unsaturated FAs contents of the CW15-24 and CW15-85 strains were 55.45% and 56.15%, which were significantly enriched compared to the wild type CW15 (48.39%). Under the optimized conditions, the content of lipid by overexpressed the ACCa gene in the mutant CW15-85 (0.46 g/l) was 1.16-fold greater than control through optimization of N and P sources. Altogether, our data clearly demonstrate that ACCa overexpression in C. reinhardtii can directly increase the synthesis of fatty acids.

Characterization of a bifunctional alginate lyase as a new member of the polysaccharide lyase family 17 from a marine strain BP-2 Abstract Objectives Bifunctional alginate lyase can efficiently saccharify alginate biomass and prepare functional oligosaccharides of alginate. Results A new BP-2 strain that produces alginate lyase was screened and identified from rotted Sargassum. A new alginate lyase, Alg17B, belonging to the polysaccharide lyase family 17, was isolated and purified from BP-2 fermentation broth by freeze-drying, dialysis, and ion exchange chromatography. The enzymatic properties of the purified lyase were investigated. The molecular weight of Alg17B was approximately 77 kDa, its optimum reaction temperature was 40–45 °C, and its optimum reaction pH was 7.5–8.0. The enzyme was relatively stable at pH 7.0–8.0, with a temperature range of 25–35 °C, and the specific activity of the purified enzyme reached 4036 U/mg. A low Na+ concentration stimulated Alg17B enzyme activity, but Ca2+, Zn2+, and other metal ions inhibited it. Substrate specificity analysis, thin-layer chromatography, and mass spectrometry showed that Alg17B is an alginate lyase that catalyses the hydrolysis of sodium alginate, polymannuronic acid (polyM) and polyguluronic acid to produce monosaccharides and low molecular weight oligosaccharides. Alg17B is also bifunctional, exhibiting both endolytic and exolytic activities toward alginate, and has a wide substrate utilization range with a preference for polyM. Conclusions Alg17B can be used to saccharify the main carbohydrate, alginate, in the ethanolic production of brown algae fuel as well as in preparing and researching oligosaccharides.

Purification, identification and characterization of an esterase with high enantioselectivity to ( S )-ethyl indoline-2-carboxylate Abstract Objective To purify an esterase which can selectively hydrolyze (R,S)-ethyl indoline-2-carboxylate to produce (S)-indoline-2-carboxylic acid and characterize its enzymatic properties. Results An intracellular esterase from Bacillus aryabhattai B8W22 was isolated and the purified protein was identified as a carboxylesterase by MALDI-TOF mass spectrometry. The enzyme (named BaCE) was 59.03-fold purification determined to be of approximately 35 kDa. Its specific activity was 0.574 U/mL with 20% yield. The enzyme showed maximum activity at pH 8.5 and 30 °C and was stable at 20–30 °C using pNPB as the substrate. The Km, Vmax, kcat and kcat/Km of the esterase were 0.52 mM, 6.39 μM/min, 26.87 min−1 and 51.67 mM/min, respectively. The esterase demonstrated high enantioselectivity toward (S)-ethyl indoline-2-carboxylate with 96.55% e.e.p at 44.39% conversion, corresponding to an E value of 133.45. Conclusions In this study, a new esterase BaCE with an apparent molecular mass of 35 kDa was purified to homogeneity for the first time. The esterase from Bacillus aryabhattai B8W22 was isolated with a purification more than 59-fold and a yield of 20% by anion exchange chromatography and hydrophobic interaction chromatography. And its biochemical characterization were described in detail with pNPB as substrate. It displayed high enantioselectivity toward (S)-ethyl indoline-2-carboxylate. We next plan to highly express esterase BaCE in Escherichia coli, and apply it to industrial production of (S)-indoline-2-carboxylic acid.

Towards a surrogate system to express human lipid binding TCRs Abstract Background Previously we reported that natural nut lipids were necessary for sensitization and that natural killer T cells (NKTs) must play a critical role in the development of food allergic responses. A major bottleneck in further understanding the interaction of nut lipids with the cells of the human immune system is the lack of well-characterized lipid responsive human cell lines. Objective In the present study, we engineered human T cell receptor (TCR) sequences TRAV10 and TRBV25 responsive to α-GalCer into a stable murine iNKT hybridoma and surrogate human T cell lines. Results The murine hybridoma system has shown to be problematic. To overcome this limitation, the expression of human TCR α/β sequences has been achieved driven by a bidirectional promoter on a plasmids or a lentivirus system, employing stable DC cell lines as lipid presenting cells, and a stable T cell line as a surrogate system. Further, a commercial human Jurkat T cell line containing an inducible secreted luciferase reporter construct was shown to be functional and can be used for a transient expression of human TCRs in a lipid screening program. The transfection efficiencies were improved using the lentivirus polycistronic constructs containing the P2A sequence in a TCR αβ/γδ null cell line (Jurkat 76). Conclusions The results suggest that the mis-pairing of the endogenous α/β TCR during ER folding in the presence of the new human TCR sequences could be impairing the functionality of the TCR lipid receptors. The surrogate systems presented here are important first steps in the establishment of human cell-specific lipid responsive libraries for the study of natural lipid substances.

Kinetics and thermodynamics of lipase catalysed synthesis of propyl caprate Abstract Objective To investigate kinetics and thermodynamics of lipase-catalyzed esterification of capric acid with 1-propyl alcohol in a solvent-free system for synthesis of propyl caprate. Results The capric acid conversion of 83.82% is achieved at temperature 60 °C, speed of agitation 300 rpm, molar ratio acid:alcohol 1:3, enzyme loading 2% (w/w) and molecular sieves loading 5% (w/w). The activation energy (Ea) for the reaction was determined as 37.79 kJ mol−1. Furthermore, enthalpy (ΔH), entropy (ΔS) and Gibbs free energy (ΔG) values were found out to be + 90.45 kJ mol−1, + 278.99 J mol−1 K−1 and − 2.35 kJ mol−1 respectively. Conclusions The results showed that the lipase-catalyzed esterification exhibits an ordered bi–bi mechanism with capric acid inhibiting the reaction and forming the dead-end complex with the lipase. Under the given set of reaction conditions, the lipase catalysed esterification reaction was anticipated to be spontaneous, referring to the value of the Gibbs free energy change (ΔG). Moreover, the esterification process was found to be endothermic, based on the values of enthalpy (ΔH) and entropy (ΔS).

Imaging aquatic animal cells and associated pathogens by atomic force microscopy in air Abstract Atomic force microscopy (AFM) is a sophisticated imaging tool with nanoscale resolution that is widely used in structural biology, cell biology, and material science, among other fields. However, to date it has rarely been applied to the study of aquatic animals, especially on one of the main cultured species, shrimp. One reason for this is that no shrimp cell line established until now, primary cell is fragile and difficult to be studied under AFM. In this study, we used AFM to image three different types of biological material from shrimp (Litopenaeus vannamei) in air, including hemocytes and two associated pathogens. Without obvious deformations when the cells were imaged in air and in the case for the haemocytes and the cells were fixed as well. The result suggests hydrophobic glass coverslips are a suitable substrate for adhesion of these samples. The method described here can be applied to the preparation of other fragile biological samples from aquatic animals for high-resolution analyses of host–pathogen interactions and other basic physiological processes.

Ac25 in Autographa californica multiple nucleopolyhedrovirus was crucial for progeny budded virion production Abstract Objectives To analyze the effect of Ac25 on the proliferation of AcMNPV (Autographa californica multicapsid nucleopolyhedrovirus) progeny virus and its function in virogenic stroma. Results AcMNPV is a model of baculovirus and is the most widely studied baculovirus. Ac25, as a single-stranded DNA-binding protein, is involved in viral genomic DNA replication. Viral proliferation assay showed that AcMNPV progeny virus could not be produced when Ac25 was knocked out, which indicated it was crucial for BV production. Absolute quantitative PCR analysis indicated that Ac25 was able to promote replication of the AcMNPV genome in host Sf9 cells. It was also found that Ac25 could increase the transcription level of 38k and vp39 late expression genes, and inhibit host cell proliferation. Conclusion Ac25 is highly accumulated in the nucleus and promotes progeny virus production by stimulating viral genome replication and up-regulating the expression of late genes. Two potential applications of vAc-Ac25-EGFP were proposed: an improved bac-to-bac eukaryotic protein expression systems and biopesticides.

Identification of internal control genes for circular RNAs Abstract Objective At present, no studies have established internal control genes for circular RNA (circRNA) analyses. We aimed to identify reference circRNAs for real-time quantitative PCR (RT-qPCR). Results After analyzing the RNA-seq data, we obtained 50 circRNAs that were expressed in all samples. We ranked these 50 circRNAs according to their stability and obtained the six most stable circRNAs. We further evaluated the stability of the six circRNAs and three linear control genes (i.e., GAPDH, β-actin and 18S rRNA) in 22 cell lines. Our results indicated that hsa_circ_0000284 (circHIPK3) and hsa_circ_0000471 (circN4BP2L2) were the two most stable genes. After removing linear RNAs or including the cells treated with Adriamycin, NH4Cl and shikonin, the two most stable genes were hsa_circ_0000471 and hsa_circ_0000284. The amplification efficiency was 100% for hsa_circ_0000471 and 95% for hsa_circ_0000284. Conclusions In conclusion, since the stability of circRNAs is higher than that of linear RNAs, hsa_circ_0000284 and hsa_circ_0000471 may be used as reference genes not only for circRNAs but also for other kinds of RNAs. The findings in the present study fill the gap of lacking reference genes in the detection of circRNAs.