Thermostability enhancement of polyethylene terephthalate degrading PETase using self- and nonself-ligating protein scaffolding approaches

Polyethylene terephthalate (PET) hydrolase enzymes show promise for enzymatic PET degradation and green recycling of single-use PET vessels representing a major source of global pollution. Their full potential can be unlocked with enzyme engineering to render activities on recalcitrant PET substrates commensurate with cost-effective recycling at scale. Thermostability is a highly desirable property in industrial enzymes, often imparting increased robustness and significantly reducing quantities required. To date, most engineered PET hydrolases show improved thermostability over their parental enzymes. Here, we report engineered thermostable variants of Ideonella sakaiensis PET hydrolase enzyme (IsPETase) developed using two scaffolding strategies. The first employed SpyCatcher-SpyTag technology to covalently cyclize IsPETase, resulting in increased thermostability that was concomitant with reduced turnover of PET substrates compared to native IsPETase. The second approach using a GFP-nanobody fusion protein (vGFP) as a scaffold yielded a construct with a melting temperature of 80°C. This was further increased to 85°C when a thermostable PETase variant (FAST PETase) was scaffolded into vGFP, the highest reported so far for an engineered PET hydrolase derived from IsPETase. Thermostability enhancement using the vGFP scaffold did not compromise activity on PET compared to IsPETase. These contrasting results highlight potential topological and dynamic constraints imposed by scaffold choice as determinants of enzyme activity.     

Gene expression studies on developing kernels of maize sucrose synthase (SuSy) mutants show evidence for a third SuSy gene

Previous studies have identified two tissue- and cell-specific, yet functionally redundant, sucrose synthase (SuSy) genes, Sh1 and Sus1, which encode biochemically similar isozymes, SH1 and SUS1 (previously referred to as SS1 and SS2, respectively). Here we report evidence for a third SuSy gene in maize, Sus3, which is more similar to dicot than to monocot SuSys. RNA and/or protein blot analyses on developing kernels and other tissues show evidence of expression of Sus3, although at the lowest steady-state levels of the three SuSy gene products and without a unique pattern of tissue specificity. Immunoblots of sh1sus1-1 embryos that are either lacking or deficient for the embryo-specific SUS1 protein have shown a protein band which we attribute to the Sus3 gene, and may contribute to the residual enzyme activity seen in embryos of the double mutant. We also studied developing seeds of the double mutant sh1sus1-1, which is missing 99.5% of SuSy enzyme activity, for evidence of co-regulation of several genes of sugar metabolism. We found a significant reduction in the steady-state levels of Miniature-1 encoded cell wall invertase2, and Sucrose transporter (Sut) mRNAs in the double mutant, relative to the lineage-related sh1Sus1 and sh1Sus1 kernels. Down-regulation of the Mn1 gene was also reflected in significant reductions in cell wall invertase activity. Co-regulatory changes were not seen in the expression of Sucrose phosphate synthase, UDP-glucose pyrophosphorylase, and ADP-glucose pyrophosphorylase.     

Analysis of DNA curvature distribution in mycobacterial promoters using theoretical models

In this paper, 125 different mycobacterial promoters are analyzed for their DNA curvature distribution using several di- and tri-nucleotide dependent models of DNA curvature. Different models give similar behavior and therefore qualitative validation of the results. Mycobacterial promoters resembling the E. coli sigma(70) type have almost 81% (85%) sequences having medium and high curvature profiles using dinucleotide-dependent models. Non-E. coli sigma(70) type mycobacterial promoters have comparatively higher percent of low curvature profiles. Very few extended -10 promoters have low curvature profiles. Mycobacterial promoters having A(n)T(m) (n+m > or =3) tract in the upstream region of -35 box and repeated in phase with each other have high curvature profiles. M. smegmatis promoters have high curvature profiles compared to M. tuberculosis promoters.     

Hyperexpression and purification of biologically active human luteinizing hormone and human chorionic gonadotropin using the methylotropic yeast, Pichia pastoris

The glycoprotein hormones, luteinizing hormone (LH), human chorionic gonadotropin (hCG), thyroid stimulating hormone (TSH), and follicle stimulating hormone (FSH), play important roles in overall physiology and reproduction. These hormones are heterodimeric molecules consisting of an identical alpha subunit non-covalently associated with the hormone-specific beta subunit. The inherent structural intricacies possessed by these hormones make them very interesting model systems for structure-function relationship studies of complex dimeric glycoproteins. The structural studies, as well as, the therapeutic applications require large quantities of biologically active hormones free of any contaminants. In this study, we report hyperexpression and purification of biologically active recombinant hLH and hCG expressed using Pichia pastoris expression system. A combination of hydrophobic interaction chromatography and ion exchange chromatography has been used to purify these recombinant hormones to homogeneity. Using a number of biochemical and immunological criteria, the recombinant hormones have been shown to be similar to the natural hormones and were equally biologically active. The preliminary data also suggested that P. pastoris cells express a low molecular weight isoform of hCG that appeared to be less glycosylated. This isoform exhibited lesser affinity for the receptor as compared to hCG, but was found to be fully biologically active.     

Synthesis of phytochelatins in vetiver grass upon lead exposure in the presence of phosphorus

In a hydroponic setting, we investigated the possible role of phytochelatins (metal-binding peptides) in the lead (Pb) tolerance of vetiver grass (Vetiveria zizanioides L.). Pb was added to the nutrient medium at concentrations ranging from 0 to 1,200 mg L^?1. Furthermore, we simulated the effect of soil phosphorus (P) on potentially plant available Pb by culturing vetiver grass in P-rich nutrient media. After 7 days of exposure to Pb, we evaluated the Pb uptake by vetiver grass. Results indicate that vetiver can accumulate Pb up to 3,000 mg kg^?1 dry weight in roots with no toxicity. Formation of lead phosphate inhibited Pb uptake by vetiver, suggesting the need for an environmentally safe chelating agent in conjunction with phytoremediation to clean up soils contaminated with lead-based paint. Unambiguous characterization of phytochelatins (PC_n) was possible using high pressure liquid chromatography coupled with electrospray ionization mass spectrometry (LC-ESMS). Vetiver shows qualitative and quantitative differences in PC_n synthesis between root and shoot. In root tissue from vetiver exposed to 1,200 mg Pb L^-1, phytochelatins ranged from PC₁ to PC₃. Collision-induced dissociation of the parent ion allowed confirmation of each PC_n based on the amino acid sequence. Possible Pb-PC₁ and Pb₂-PC₁ complexes were reported in vetiver root at the highest Pb concentration. The data from these experiments show that the most probable mechanism for Pb detoxification in vetiver is by synthesizing PC_n and forming Pb–PC_n complexes.     

Antihyperglycemic, antistress and nootropic activity of roots of Rubia cordifolia Linn

Effect of alcoholic extract of roots of Rubia cordifolia was studied on elevated blood glucose level in alloxan treated animals. The extract reduced the blood sugar level raised by alloxan. Effect of alcoholic extract was also investigated on cold restraint induced stress and on scopolamine-induced memory impairment. Alcoholic extract enhanced brain gamma-amino-n-butyric acid (GABA) levels and decreased brain dopamine and plasma corticosterone levels. Acidity and ulcers caused due to cold restraint stress were inhibited by alcoholic extract. Animals treated with alcoholic extract spent more time in open arm in elevated plus maze model. It also antagonized scopolamine induced learning and memory impairment. Baclofen induced catatonia was potentiated by alcoholic extract.