ABSTRACT
The nature of amino acid residue 47 shows a clear discrimination between the different groups of cyclodextrin glycosyltransferase (CGTase). The effects of amino acid side chain at position 47 on cyclodextrin product specificity were investigated by replacing Lys47 in the CGTase from Paenibacillus macerans strain JFB05-01 with arginine, histidine, threonine, serine, or leucine. All of the mutations reduced alpha-cyclodextrin-forming activity, whereas significant increases in beta-cyclodextrin-forming activity were achieved. Especially, the mutations of Lys47 into threonine, serine, or leucine converted P. macerans CGTase from alpha-type to beta/alpha-type. As a result, all of the mutants displayed a shift in product specificity toward the production of beta-cyclodextrin. Thus, they were more suitable for the industrial production of beta-cyclodextrin than the wild-type enzyme. The enhancement of beta-cyclodextrin specificity might be due to weakening or removal of hydrogen-bonding interactions between the side chain of residue 47 and the bent intermediate specific for alpha-cyclodextrin formation.
Subject(s)
Glucosyltransferases/chemistry , Lysine/genetics , Mutagenesis, Site-Directed , Paenibacillus/enzymology , beta-Cyclodextrins/metabolism , Glucosyltransferases/genetics , Leucine/chemistry , Lysine/chemistry , Serine/chemistry , Structure-Activity Relationship , Substrate Specificity , Threonine/chemistry , alpha-Cyclodextrins/metabolismABSTRACT
Overexpression of recombinant genes in Escherichia coli and targeting recombinant proteins to the culture medium are highly desirable for the production of industrial enzymes. However, a major barrier is inadequate secretion of recombinant protein across the two membranes of E. coli cells. In the present study, we have attempted to circumvent this secretion problem of the recombinant alpha-cyclodextrin glycosyltransferase (alpha-CGTase) from Paenibacillus macerans strain JFB05-01. It was found that glycine, as a medium supplement, could enhance the extracellular secretion of recombinant alpha-CGTase in E. coli. In the culture with glycine at the optimal concentration of 150 mM, the alpha-CGTase activity in the culture medium reached 23.5 U/mL at 40 h of culture, which was 11-fold higher than that of the culture in regular TB medium. A 2.3-fold increase in the maximum extracellular productivity of recombinant alpha-CGTase was also observed. However, further analysis indicated that glycine supplementation exerted impaired cell growth as demonstrated by reduced cell number and viability, increased cell lysis, and damaged cell morphology, which prevented further improvement in overall enzyme productivity. Significantly, Ca(2+) could remedy cell growth inhibition induced by glycine, thereby leading to further increase in the glycine-enhanced extracellular secretion of recombinant alpha-CGTase. In the culture with 150 mM glycine and 20 mM Ca(2+), both extracellular activity and maximum productivity of recombinant enzyme were 1.5-fold higher than those in the culture with glycine alone. To the best of our knowledge, this is the first article about the synergistic promoting effects of glycine and Ca(2+) on the extracellular secretion of a recombinant protein in E. coli.
Subject(s)
Calcium/pharmacology , Escherichia coli/drug effects , Escherichia coli/metabolism , Glucosyltransferases/metabolism , Glycine/pharmacology , Recombinant Proteins/metabolism , Bacteria/enzymology , Cell Membrane Permeability/drug effects , Drug Synergism , Escherichia coli/growth & development , Gene Expression , Glucosyltransferases/biosynthesis , Glucosyltransferases/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/geneticsABSTRACT
To achieve detection, monitoring and automation of starch graft copolymerzation, the investigation was carried out according to the luminescence of free radical produced in the reaction. The investigation on the fluorescence spectra of starch-water suspension excited by ultraviolet light and its characteristics contributes to the study of the characteristics and mechanisms of free radical coming into being, emitting and disappearing. The experimental results indicate that starch-water suspension can emit fluorescence, whose peak wavelength is about 339 nm. Furthermore, the physical mechanism of fluorescence emission of starch is analyzed. It is considered that this fluorescence comes from the transition from nonbonding electrons in the hetero-atom (O) of the functional group (C--O--C) called ether linkage to the antibonding orbital sigma*.