Transgalactosylation in a Water-Solvent Biphasic Reaction System with β-Galactosidase Displayed on the Surfaces of Bacillus subtilis Spores

ABSTRACT The ever-increasing industrial demand for biocatalysis necessitates innovations in the preparation and stabilization of biocatalysts. In this study, we demonstrated that β-galactosidase (β-Gal) displayed on Bacillus spores by fusion to the spore coat proteins (CotG) may be used as a whole-cell immobilized biocatalyst for transgalactosylation in water-solvent biphasic reaction systems. The resulting spores had a specific hydrolytic activity of 5 × 103 U/g (dry weight) of spores. The β-Gal was tightly attached to the spore surface and was more stable in the presence of various organic solvents than its native form was. The thermostability of the spore-displayed enzyme was also increased, and the enzyme was further stabilized by chemically cross-linking it with glutaraldehyde. With spore-displayed β-Gal, octyl-β-d-galactopyranoside was synthesized at concentrations up to 27.7 mM (8.1 g/liter) with a conversion yield of 27.7% (wt/wt) after 24 h from 100 mM lactose and 100 mM octanol dissolved in phosphate buffer and ethyl ether, respectively. Interestingly, the spores were found to partition mainly at the interface between the water and solvent phases, and they were more available to catalysis between the two phases, as determined by light microscopy and confocal fluorescence microscopy. We propose that spore display not only offers a new and facile way to construct robust biocatalysts but also provides a novel basis for phase transfer biocatalytic processes.

Download Full-text

Production of Furfural from Process-Relevant Biomass-Derived Pentoses in a Biphasic Reaction System

ACS Sustainable Chemistry & Engineering ◽

10.1021/acssuschemeng.7b00215 ◽

2017 ◽

Vol 5 (7) ◽

pp. 5694-5701 ◽

Cited By ~ 61

Author(s):

Ashutosh Mittal ◽

Stuart K. Black ◽

Todd B. Vinzant ◽

Marykate O’Brien ◽

Melvin P. Tucker ◽

...

Keyword(s):

Reaction System ◽

Biphasic Reaction

Download Full-text

SpoVID Guides SafA to the Spore Coat inBacillus subtilis

Journal of Bacteriology ◽

10.1128/jb.183.10.3041-3049.2001 ◽

2001 ◽

Vol 183 (10) ◽

pp. 3041-3049 ◽

Cited By ~ 56

Author(s):

Amanda J. Ozin ◽

Craig S. Samford ◽

Adriano O. Henriques ◽

Charles P. Moran

Keyword(s):

Direct Interaction ◽

Spore Coat ◽

Coat Proteins ◽

Yeast Two Hybrid System ◽

Spore Coat Proteins ◽

Basement Layer ◽

Two Hybrid ◽

Spore Coat Protein

ABSTRACT Bacteria assemble complex structures by targeting proteins to specific subcellular locations. The protein coat that encasesBacillus subtilis spores is an example of a structure that requires coordinated targeting and assembly of more than 24 polypeptides. The earliest stages of coat assembly require the action of three morphogenetic proteins: SpoIVA, CotE, and SpoVID. In the first steps, a basement layer of SpoIVA forms around the surface of the forespore, guiding the subsequent positioning of a ring of CotE protein about 75 nm from the forespore surface. SpoVID localizes near the forespore membrane where it functions to maintain the integrity of the CotE ring and to anchor the nascent coat to the underlying spore structures. However, it is not known which spore coat proteins interact directly with SpoVID. In this study we examined the interaction between SpoVID and another spore coat protein, SafA, in vivo using the yeast two-hybrid system and in vitro. We found evidence that SpoVID and SafA directly interact and that SafA interacts with itself. Immunofluorescence microscopy showed that SafA localized around the forespore early during coat assembly and that this localization of SafA was dependent on SpoVID. Moreover, targeting of SafA to the forespore was also dependent on SpoIVA, as was targeting of SpoVID to the forespore. We suggest that the localization of SafA to the spore coat requires direct interaction with SpoVID.

Download Full-text

Efficient microwave-assisted production of furanics and hydrochar from bamboo (Phyllostachys nigra “Boryana”) in a biphasic reaction system: effect of inorganic salts

Biomass Conversion and Biorefinery ◽

10.1007/s13399-021-01372-6 ◽

2021 ◽

Author(s):

Nick Sweygers ◽

Mohammadreza Kamali ◽

Tejraj M. Aminabhavi ◽

Raf Dewil ◽

Lise Appels

Keyword(s):

Reaction System ◽

Inorganic Salts ◽

System Effect ◽

Microwave Assisted ◽

Biphasic Reaction ◽

Phyllostachys Nigra

Download Full-text

Cloning and expression of a cDNA that comprises part of the gene coding for a spore coat protein of Dictyostelium discoideum

Molecular and Cellular Biology ◽

10.1128/mcb.4.11.2273-2278.1984 ◽

1984 ◽

Vol 4 (11) ◽

pp. 2273-2278

Author(s):

B C Dowds ◽

W F Loomis

Keyword(s):

Dictyostelium Discoideum ◽

Cdna Clone ◽

Single Gene ◽

Polyclonal Antiserum ◽

Cloning And Expression ◽

Spore Coat ◽

Coat Proteins ◽

Developmentally Regulated ◽

Gene Coding ◽

Spore Coat Proteins

The three major spore coat proteins of Dictyostelium discoideum are developmentally regulated, cell-type-specific proteins. They are packaged in prespore vesicles and then secreted to form the outer layer of spore coats. We have isolated a cDNA clone from the gene coding for one of these proteins, SP96, a glycoprotein of 96,000 daltons. We screened the cDNA bank by the method of hybrid select translation followed by immunoprecipitation of the translation products with SP96-specific polyclonal antiserum. We found that the gene was first transcribed into stable mRNA a few hours before the time of detection of SP96 synthesis and that the mRNA, like the protein, accumulated specifically in prespore cells and spores. SP96 constituted the same proportion of newly synthesized protein as the proportion of its message in polyadenylated RNA. SP96 appeared to be encoded by a single gene as judged by Southern blot analysis of digested genomic DNA hybridized to the cDNA clone.

Download Full-text

Identification of proteins in the exosporium of Bacillus anthracis

Microbiology ◽

10.1099/mic.0.26681-0 ◽

2004 ◽

Vol 150 (2) ◽

pp. 355-363 ◽

Cited By ~ 160

Author(s):

Caroline Redmond ◽

Leslie W. J. Baillie ◽

Stephen Hibbs ◽

Arthur J. G. Moir ◽

Anne Moir

Keyword(s):

Bacillus Anthracis ◽

Amino Acid Sequences ◽

Coat Proteins ◽

Wild Type ◽

Sds Page ◽

Adsorbed Proteins ◽

Spore Coat Proteins ◽

Ames Strain ◽

A Site ◽

Novel Protein

Spores of Bacillus anthracis, the causative agent of anthrax, possess an exosporium. As the outer surface layer of these mature spores, the exosporium represents the primary contact surface between the spore and environment/host and is a site of spore antigens. The exosporium was isolated from the endospores of the B. anthracis wild-type Ames strain, from a derivative of the Ames strain cured of plasmid pXO2−, and from a previously isolated pXO1−, pXO2− doubly cured strain, B. anthracis UM23Cl2. The protein profiles of SDS-PAGE-separated exosporium extracts were similar for all three. This suggests that avirulent variants lacking either or both plasmids are realistic models for studying the exosporium from spores of B. anthracis. A number of loosely adsorbed proteins were identified from amino acid sequences determined by either nanospray-MS/MS or N-terminal sequencing. Salt and detergent washing of the exosporium fragments removed these and revealed proteins that are likely to represent structural/integral exosporium proteins. Seven proteins were identified in washed exosporium: alanine racemase, inosine hydrolase, ExsF, CotY, ExsY, CotB and a novel protein, named ExsK. CotY, ExsY and CotB are homologues of Bacillus subtilis outer spore coat proteins, but ExsF and ExsK are specific to B. anthracis and other members of the Bacillus cereus group.

Download Full-text

Dictyostelium prespore-specific gene Dp87 encodes a sorus matrix protein

Journal of Cell Science ◽

10.1242/jcs.107.3.397 ◽

1994 ◽

Vol 107 (3) ◽

pp. 397-403 ◽

Cited By ~ 1

Author(s):

H. Nakao ◽

A. Yamamoto ◽

I. Takeuchi ◽

M. Tasaka

Keyword(s):

Matrix Protein ◽

Specific Protein ◽

Specific Gene ◽

Coat Proteins ◽

Fruiting Bodies ◽

Protein Fragment ◽

Microscopic Studies ◽

Spore Coat Proteins ◽

Space Matrix ◽

Membranous Material

In this paper we report on the characteristics of the product of a prespore-specific gene (Dp87) of Dictyostelium discoideum. Polyclonal antibody was made against a bacterially synthesized Dp87-encoded protein fragment. Using this antibody, the product was characterized by immunochemical and immunocytological methods. It was shown that the Dp87-encoded protein is a prespore-specific protein with a molecular mass of 83 kDa, which first appears at the standing slug stage and persists in mature fruiting bodies. Western blot studies revealed the presence of an additional 81 kDa protein prior to the appearance of the 83 kDa protein from the tipped aggregate to the standing slug stage, thus indicating the former to be a precursor protein. Immunocytochemical and immunoelectron microscopic studies showed that the protein is bound to ER at the early stages of development when only the 81 kDa protein is present. At the later stages when the 83 kDa protein predominates, however, it becomes localized in prespore-specific vacuoles (PSVs) and is associated with the inner fibrous material of PSVs, but not with the peripheral membranous material. This is in contrast to spore coat proteins, which are localized in PSVs from the beginning of their appearance and associated with both structures of PSVs. In mature fruiting bodies, most Dp87 protein is localized to the interspore space (matrix) of the sori, with some left on the surface of the stalk tube. Disruptants of the Dp87 gene were also produced. Although they contained neither 81 kDa nor 83 kDa protein, they showed no phenotypic defects as compared to the parental strain.

Download Full-text

Impairment of transalveolar fluid transport and lung Na+-K+-ATPase function by hypoxia in rats

Journal of Applied Physiology ◽

10.1152/jappl.1999.87.3.962 ◽

1999 ◽

Vol 87 (3) ◽

pp. 962-968 ◽

Cited By ~ 45

Author(s):

Satoshi Suzuki ◽

Masafumi Noda ◽

Makoto Sugita ◽

Sadafumi Ono ◽

Kaoru Koike ◽

...

Keyword(s):

Fluid Transport ◽

Weight Ratio ◽

Dry Weight ◽

Hydrolytic Activity ◽

Hypoxic Exposure ◽

Blue Dye ◽

Alveolar Type ◽

Alveolar Fluid Clearance ◽

Exposure In Vivo

We examined whether hypoxic exposure in vivo would influence transalveolar fluid transport in rats. We found a significant decrease in alveolar fluid clearance of the rats exposed to 10% oxygen for 48 h. Terbutaline did not stimulate alveolar fluid clearance, and alveolar fluid cAMP levels were lower than those determined in normoxia experiment. Hypoxia did not influence the alveolar fluid lactate dehydrogenase levels, Evans blue dye fluid-to-serum concentration ratio, or lung wet-to-dry weight ratio, indicating no significant change in the permeability of alveolar-capillary barrier. Histological examination showed no significant fluid accumulation into the interstitium and the alveolar space. Hypoxia did not reduce lung ATP content; however, we found significant decrease in Na+-K+-ATPase hydrolytic activity in lung tissue preparations and isolated alveolar type II cells. Our data indicate that hypoxic exposure in vivo impairs transalveolar fluid transport, and this impairment is related to the decrease in alveolar epithelial Na+-K+-ATPase hydrolytic activity but is not secondary to the alteration of cellular energy source.

Download Full-text