Xing-hua Li

Enhanced cellulase production of the Trichoderma viride mutated by microwave and ultraviolet

Cellulase-producing fungi Trichoderma viride were cultured and fermented on the solid-state wheat bran fermentation medium. The characteristics of its carboxymethyl cellulase (CMCase) in the condition of this solid-state fermentation were evaluated, and the optimum culture time, optimum pH and optimum temperature for CMCase activity of T. viride fermented in this solid state were 60h, 5.0 and 50 degrees C, respectively. Carboxymethyl cellulose sodium (CMC-Na) and Congo red were used to screen the strains that had stronger ability to produce enzymes. After the compound mutagenesis by microwave and ultraviolet, seven mutant strains (M-B1-M-B7) were selected and their CMCase activities were assayed. Five of them (M-B1, M-B2, M-B3, M-B5 and M-B7) had significantly stronger ability to produce enzymes than the normal wild type, and they were also very stable for a long period up to 9 generations to produce cellulase. Molecular studies showed that there were some base mutations in endoglucanase I (EG I) genes of mutants M-B1, M-B2, M-B3 and M-B5, but no change in M-B7, suggesting that some amino mutations in EG I proteins caused by base mutations could lead to enhanced cellulase production.

The most stirring technology in future: cellulase enzyme and biomass utilization.

In recent years, fundamental and applied researches on cellulase enzyme have not only generated significant scientific knowledge but also have revealed their enormous potential in biotechnology. Growing attention has been devoted to its bioconversion of biomass into fuel ethanol, considered the cleanest liquid fuel alternative to fossil fuels. Significant advances have been made towards the production and alteration technology of cellulase enzyme. This review simply introduces cellulose and cellulase enzyme, gives a broad overview of the current research status of cellulase enzyme, briefly refers to its applied fields, and lastly summarizes its promising prospects.

Anti-oxidation and immune responses in mice upon exposure to manganese superoxide dismutase expressed in silkworm larvae, Bombyx mori L.

With manganese superoxide dismutase expressed in silkworm larvae, Bomby mori L, we investigate the effects of silkworm larvae powder containing SOD on the antioxidation and the immune system of mouse. The contents of MDA both in mice plasma or liver organ treated with silkworm larvae powder containing manganese superoxide dismutase were reduced compare to control. The superoxide dismutase (SOD) and glutathione peroxidase (GSH‐Px) activities both in plasma or liver organ of the treated mice were significantly higher than that of both control and bromobenzene treated mice (group‐BM), suggesting the silkworm larvae powder containing SOD play a positive role in anti‐oxidation in mice. This experiment was also designed to investigate the effects of silkworm larvae powder containing SOD on the immune system of mouse, focused on hemolysin response, hemagglutination against SRBC and the activity of natural killer (NK) cells. All treated mice showed significant increase in hemolysin response to SRBC and demonstrated an activation of NK cell function by the SOD‐contained silkworm larvae powder, which suggest a promotion in humoral immunity. The results suggested the SOD expressed in silkworm maybe have potential application in medicine.

Cloning and expression pattern of 3-dehydroecdysone 3β-reductase (3DE 3β-reductase) from the silkworm, Bombyx mori L.

Molting in insects is regulated by molting hormones (ecdysteroids), which are also crucial to insect growth, development, reproduction, etc. Ecdysone was inactivated to 3-dehydroecdysone (3DE) under ecdysone oxidase (EO), and followed by NAD(P)H-dependent irreversible reduction to 3-epiecdysteroid under 3DE 3a-reductase. On the other hand, 3-dehydroecdysone undergoes reversible reduction to ecdysone by 3DE 3β-reductase in the hemolymph. In this article, we cloned and characterized 3-dehydroecdysone 3β-reductase (3DE 3β-reductase) in the different tissues and the developing stage from the silkworm, Bombyx mori L. The B. mori 3DE 3β-reductase cDNA contains an ORF 972 bp and the deduced protein sequence containing 323 amino acid residues. Analysis showed that the deduced 3DE 3β-reductase belongs to the aldo-keto reductase (AKR) superfamily, which has the NAD(P)-binding domain, indicating that the function of 3DE 3β-reductase depends on the existence of NAD(P)H. Using Escherichia coli, a high level expression of a fusion polypeptide band of approx. 40 kDa was observed. The high transcription of 3DE 3β-reductase was mainly observed in the genitalia and fatty bodies in the third day of the fifth-instar larvae, followed next in the head, epidermis, and hemocytes. The expression of 3DE 3β-reductase in the early of every instar was lower than that in the late of instar. When the titer of 3DE is low, higher expression of 3DE 3β-reductase is necessary to maintain the ecdysone titer in body through converting 3DE to ecdysone, while the 3DE titer is high, the expression of 3DE 3β-reductase showed feedback inhibition.

Cloning and characterization of the Bombyx mori ecdysone oxidase

The physiological titer of molting hormones in insects depends on relative activities of synthesis and degradation pathways. Ecdysone oxidase (EO) is a key enzyme in the inactivation of ecdysteroid. However, there are only a few reports on ecdysteroid inactivation and its enzymes in silkworm. In this study, we cloned and characterized the Bombyx mori EO (BmEO). The BmEO cDNA contains an ORF of 1,695 bp and the deduced protein sequence contains 564 amino acid residues. The deduced protein sequence contains two functional domains of glucose-methanol-choline oxidoreductase in N-terminal and C-terminal. Comparing the expression levels of BmEO in different tissues, high transcription was mainly present in hemocytes. Reduced expression of this enzyme is expected to lead to pathological accumulation of ecdysone in the hemolymph of silkworm larvae or pupae. Our data show that RNA inference of BmEO transcripts resulted in the accumulation of ecdysteroid and death of larvae or pupae. We infer that EO is a crucial element in the physiology of insect development.

Proteomic identification of the silkworm (Bombyx mori L) prothoracic glands during the fifth instar stage.

Although the ecdysteroid of the silkworm had been studied for decades, the proteome of the prothoracic gland, the primary source of ecdysteroid hormones, has not been studied previously. In the present paper, we utilized a proteomic approach to investigate the fifth instar prothoracic gland during the growth and development of the silkworm, Bombyx mori L. The two-dimensional electrophoresis results showed that the majority of proteins were acidic proteins, especially concentrated in the area of 25-65 kDa, with pI values of between 4 and 7, and the difference was not distinct. When compared with Qiufeng (Japanese strain), the interspecific distinction was larger than the intraspecific distinction, and 19 particular spots, excized from the third, fifth and ninth days of p50 (Chinese strain) and Qiufeng were subjected to MALDI-TOF-MS (matrix-assisted laser-desorption ionization-time-of-flight MS) analysis. We sorted them into seven catagories: energetics and/or metabolism, storage proteins, protection, lipid metabolism, signal transduction, cell function and unknown function proteins. Of these proteins, arginine methyltransferase is discussed as playing an important role in regulating the activation of ecdysteroidogenesis via transcription or translation.

Identification and characterization of two chitin-binding proteins from the peritrophic membrane of the silkworm, Bombyx mori L.

The peritrophic membrane (PM) is a semi-permeable lining of the insect midgut, broadly analogous to the mucous lining of vertebrate gut. The PM proteins are important achievements for the function of the PM. In this study, two chitin-binding proteins (BmPM-P43 and BmPM-P41) from the PM of the silkworm, Bombyx mori, were identified and cloned. These proteins showed the molecular mass of 43 and 41 kDa, respectively. The deduced amino acid sequences codes for a protein of 381 amino acid residues and 364 amino acid residues, containing 12 and 14 cysteine residues followed by similar domain, both of them have 5 cysteine residues in similar position in the C-terminal. The confirmation of these proteins was performed by western blot analysis of recombinant BmPM-P43 and BmPM-P41. The chitin-binding activity analysis showed that the BmPM-P43 and BmPM-P41 could bind to chitin strongly. It is concluded that BmPM-P43 and BmPM-P41 contains a polysaccharide deacetylase domain instead of peritrophin domain, indicated that these two proteins may belong to a new chitin-binding protein family.

Improvement of recombinant baculovirus infection efficiency to express manganese superoxide dismutase in silkworm larvae through dual promoters of Pph and Pp10

The silkworm, Bombyx mori, has been used as an important bioreactor for the production of recombinant proteins through baculovirus expression system (BES). There are several problems which will probably be the bottleneck for practical and industrial utilization of silkworm bioreactor. Traditionally, the recombinant virus should infect the larvae through individual dorsal injection by a syringe. This is a time- and labor-consuming procedure. This drawback has become a bottleneck for practical and industrial utilization of baculovirus expression system in the silkworm bioreactor. In this paper, we constructed a dual expression baculovirus to express the renovated polyhedron and target manganese superoxide dismutase (SOD) gene under P10 and polyhedron promoters, respectively, through oral infection. The results showed that the direct injection of recombinant rBacmid/BmNPV/SOD DNA with cellfectin reagent infected the silkworm larvae partially. When next batches of larvae were fed orally with hemolymph, which was collected from first batch of injected and infected larvae, the obvious symptom of infection was found and high target SOD was expressed. These results imply it is feasible to express target genes through combination of recombinant bacmid DNA injection and oral feeding by a dual expression bacmid baculovirus.

Demonstration of protein absorption in the intestinal epithelium of fish and mice by laser scanning confocal microscopy

Abstract Selective permeability for small proteins and oligopeptides occurs in the intestinal epithelium of many animal species and humans. Whole proteins are sometimes endocytosed and undergo partial hydrolysis in intestinal epithelial cells with the probable release of essential oligopeptides into the bloodstream. Increased permeability to certain proteins can cause asthma and other metabolic disorders. Permeable proteins have also been successfully used to deliver vaccines or drugs via oral consumption. Protein absorption has been inferred in many cases and demonstrated in some cases by histochemical, tracer, and analytical techniques. However, the nature and importance of protein absorption remains largely unknown. Here, we demonstrate the movement of two lumenal proteins (GFP: 26 kDa and OFP: 23 kDa) across the intestinal epithelium of fish and mice using laser scanning confocal microscopy. The results provide evidence that small proteins can be taken up intact by intestinal epithelial cells, even though large proteins are digested to single amino acids or protein fragments before they are absorbed. Our results suggest that it is possible to orally administer some small proteinous medicines for therapeutic purposes.

Application of proteomic technology in silkworm research

Abstract From structural genome to functional one, the proteomic research has been an important content in the post-genome times. This paper presents the concept of proteome, and introduces its research technology and status in silkworm research.