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Lignin, a common natural polymers, is abundant and complex, and termites can break down and utilize the lignin in their food. In this study an attempt was made to isolate and characterize the lignolytic bacteria from termite (Reticulitermes chinensis Snyder) gut. Two strains (PY12 and MX5) with high lignin peroxidase (LiP) activity were screened using the azure B method. By analyzing their 16S rRNA, the strain PY12 was classified as Enterobacter hormaechei; MX5, as Bacillus licheniformis. We then optimized the different conditions of liquid fermentation medium, and obtained LiP activities of 278 U/L and 256 U/L for PY12 and MX5, respectively. Subsequently, we confirmed the LiP activities of the strains by evaluating their decolorizing effects on various dyes. Finally, we cloned the LiP gene of strain PY12 and successfully transferred it to Lactococcus lactis. We believe that our results provide the theoretical and practical basis for the production of genetically engineered bacteria that produce LiP, thus allowing for the utilization of naturally available lignin as an energy resource., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The gene encoding the Enterobacter amnigenus AR-37 arylsulfate sulfotransferase (ASST) was cloned, sequenced, and expressed in Escherichia coli NM522. Sequencing led to the identification of three contiguous open reading frames (ORFs) on the same strand. Based on amino acid sequence homology, ORF1, ORF2, and ORF3 are designated astA, dsbA, and dsbB, respectively. A multiple sequence alignment revealed conserved regions in ASST. An N-terminal amino acid sequence analysis of the purified ASST from E. coli NM522 (pEAST72) showed that it is subject to N-terminal processing. The specific activity of purified ASST is 436.5 U/mg of protein. The enzyme is a monomeric protein with a molecular mass of 64 kDa. Using phenol as an acceptor substrate, 4-methylumbelliferyl sulfate is the best donor substrate, followed by beta-naphthyl sulfate, p-nitrophenyl sulfate (PNS), and alpha-naphthyl sulfate. For PNS, alpha-naphthol is the best acceptor substrate, followed by phenol, resorcinol, p-acetaminophen, tyramine, and tyrosine. The enzyme has a different acceptor specificity than the enzyme purified from Eubacterium A-44. It is similar to Klebsiella K-36 and Haemophilus K-12. The apparent K(m) values for PNS using phenol as an acceptor and for phenol using PNS as a donor are 0.163 and 0.314 mM, respectively. The pI and optimum pH are 6.1 and 9.0, respectively., (Copyright 1999 Academic Press.)

beta-Lactamase from Enterobacter cloacae P99 was immobilized onto Sepharose by the cyanogen bromide activation method and the properties of the Sepharose-bound enzyme were compared with those of soluble and cell-bound enzyme. The immobilized beta-lactamase showed enhanced stability to storage at 4 degrees C (approximately 1 year) in respect to the free enzyme in solution (few days). The optimum pH for activity is similar for both Sepharose- and cell-bound beta-lactamase and extends over a broader pH range (pH 6-9) than the soluble enzyme (pH 8-9). Immobilization leads also to significant enhancement of thermal stability. Effective enzyme inhibition by flucloxacillin occurs with both soluble and Sepharose-bound beta-lactamase, whereas the cell-bound enzyme is much less (10(-5) times) inhibited. These results indicate that immobilized beta-lactamase could be usefully employed as a tool for investigating the properties of newly designed beta-lactamase inhibitors.

Predominant enterobacteria from infants with necrotizing enterocolitis (NEC) were examined for an unusual ability to ferment lactose. One such isolate, a Klebsiella pneumoniae strain, was partially induced for lactose operon expression in tryptone containing media, and was also pathogenic in a rabbit ileal loop model for NEC. A spontaneous segregant of this strain was no longer partially induced for lactose operon expression, and was no longer pathogenic in the rabbit model. The gene responsible for this phenotype was cloned. The resulting plasmid was shown to cause both partially induced lactose operon expression and pathogenicity when introduced into a laboratory K. pneumoniae strain. A K. pneumoniae mutant deficient in lactose repressor synthesis was also pathogenic in the rabbit model. These results and previous studies on the intraluminal biochemistry of infants with NEC support the hypothesis that an increased ability for lactose fermentation may be a bacterial pathogenic trait with respect to NEC.