Chitosanase from Streptomyces coelicolor A3(2): biochemical properties and role in protection against antibacterial effect of chitosan

Chitosan, an N-deacetylated derivative of chitin, has attracted much attention as an antimicrobial agent against fungi, bacteria, and viruses. Chitosanases, the glycoside hydrolases responsible for chitosan depolymerisation, are intensively studied as tools for biotechnological transformation of chitosan. The chitosanase CsnA (SCO0677) from Streptomyces coelicolor A3(2) was purified and characterized. CsnA belongs to the GH46 family of glycoside hydrolases. However, it is secreted efficiently by the Tat translocation pathway despite its similarity to the well-studied chitosanase from Streptomyces sp. N174 (CsnN174), which is preferentially secreted through the Sec pathway. Melting point determination, however, revealed substantial differences between these chitosanases, both in the absence and in the presence of chitosan. We further assessed the role of CsnA as a potential protective enzyme against the antimicrobial effect of chitosan. A Streptomyces lividans TK24 strain in which the csnA gene was inactivated by gene disruption was more sensitive to chitosan than the wild-type strain or a chitosanase-overproducing strain. This is the first genetic evidence for the involvement of chitosanases in the protection of bacteria against the antimicrobial effect of chitosan.     

Cloning and characterization of a chitosanase gene from the koji mold Aspergillus oryzae strain IAM 2660

A genomic copy of the gene coding for chitosanase (csnA) was isolated from Aspergillus oryzae IAM 2660. A. oryzae csnA contains an open reading frame that encodes a polypeptide of 245 amino acids with a calculated molecular mass of 26,500 Da. The deduced amino acid sequence of A. oryzae csnA indicates extensive similarities to those of other fungal chitosanases.     

Cloning and heterologous expression of a beta-D-mannosidase (EC 3.2.1.25)-encoding gene from Thermobifida fusca TM51

Thermobifida fusca TM51, a thermophilic actinomycete isolated from composted horse manure, was found to produce a number of lignocellulose-degrading hydrolases, including endoglucanases, exoglucanases, endoxylanases, beta-xylosidases, endomannanases, and beta-mannosidases, when grown on cellulose or hemicellulose as carbon sources. beta-Mannosidases (EC 3.2.1.25), although contributing to the hydrolysis of hemicellulose fractions, such as galacto-mannans, constitute a lesser-known group of the lytic enzyme systems due to their low representation in the proteins secreted by hemicellulolytic microorganisms. An expression library of T. fusca, prepared in Streptomyces lividans TK24, was screened for beta-mannosidase activity to clone genes coding for mannosidases. One positive clone was identified, and a beta-mannosidase-encoding gene (manB) was isolated. Sequence analysis of the deduced amino acid sequence of the putative ManB protein revealed substantial similarity to known mannosidases in family 2 of the glycosyl hydrolase enzymes. The calculated molecular mass of the predicted protein was 94 kDa, with an estimated pI of 4.87. S. lividans was used as heterologous expression host for the putative beta-mannosidase gene of T. fusca. The purified gene product obtained from the culture filtrate of S. lividans was then subjected to more-detailed biochemical analysis. Temperature and pH optima of the recombinant enzyme were 53 degrees C and 7.17, respectively. Substrate specificity tests revealed that the enzyme exerts only beta-D-mannosidase activity. Its kinetic parameters, determined on para-nitrophenyl beta-D-mannopyranoside (pNP-betaM) substrate were as follows: K(m) = 180 micro M and V(max) = 5.96 micro mol min(-1) mg(-1); the inhibition constant for mannose was K(i) = 5.5 mM. Glucono-lacton had no effect on the enzyme activity. A moderate trans-glycosidase activity was also observed when the enzyme was incubated in the presence of pNP-alphaM and pNP-betaM; under these conditions mannosyl groups were transferred by the enzyme from pNP-betaM to pNP-alphaM resulting in the synthesis of small amounts (1 to 2%) of disaccharides.     

腐皮镰孢菌壳聚糖酶的酶学性质研究及其在酿酒酵母工业菌株中的表达

摘要：【目的】本研究旨在了解腐皮镰孢菌（Fusarium solani）壳聚糖酶的基本酶学性质及其在壳寡糖生产中的应用,构建能高效分泌表达壳聚糖酶的酿酒酵母工业菌株。【方法】采用RT-PCR扩增腐皮镰孢菌壳聚糖酶的cDNA序列;通过组氨酸标签,纯化得到E. coli表达的重组壳聚糖酶,并进行基本酶学性质研究;以薄层层析、高效液相色谱等技术对该酶的酶解产物进行分析;通过马克斯克鲁维酵母（Kluyveromyces marxianus）菊粉酶信号肽（INU1A）实现壳聚糖酶在酿酒酵母工业菌株N-27中的分泌表     

Cloning and nucleotide sequence of celA1, and endo-beta-1,4-glucanase-encoding gene from Streptomyces halstedii JM8.

The celA1 gene encoding an endo-beta-1,4-glucanase from a mesophilic actinomycete, strain JM8, identified as Streptomyces halstedii, was cloned and expressed in S. lividans JI66. From the nucleotide sequence of a 1.7-kb DNA fragment we identified an open reading frame of 963 nucleotides encoding a protein of 321 amino acids, starting at TTG (instead of ATG). The Cel1 mature enzyme is a protein of 294 amino acids (after signal peptide cleavage) and can be included in the beta-glycanase family B (N. R. Gilkes, B. Henrissat, D. G. Kilburn, R. C. Miller, Jr., and R. A. J. Warren, Microbiol. Rev. 55:303-315, 1991). The Cel1 enzyme lacks a cellulose-binding domain as predicted by computer analysis of the sequence and confirmed by Avicel binding experiments. The promoter region of celA1 was identified by S1 mapping; the -35 region closely resembles those of housekeeping Streptomyces promoters. Three imperfectly repeated sequences of 15, 15, and 14 nucleotides were found upstream from celA1 [ATTGGGACCGCTTCC-(N85)-ATTGGGACCGCTTCC-(N2)-TGGGAGC GCTCCCA]; The 14-nucleotide sequence has a perfect palindrome identical to that found in several cellulase-encoding genes from Thermomonospora fusca, an alkalophilic Streptomyces strain, and Streptomyces lividans. This sequence has been implicated in the mechanism of induction exerted by cellobiose. Using an internal celA1 probe, we detected similar genes in several other Streptomyces species, most of them cellulase producers.     

Increasing the efficiency of heterologous promoters in actinomycetes

An Escherichia coli-actinomycete shuttle vector, pCJW93, was constructed which places cloned genes under the control of the thiostrepton-inducible tip promoter from Streptomyces lividans. We also constructed expression vectors bearing the actII-ORF4/PactI activator-promoter system of the actinorhodin biosynthetic pathway of Streptomyces coelicolor. With both types of vector, levels of expression varied widely in different actinomycete strains, indicating different levels of the host factors needed for optimal expression. Deletion of the actII-ORF4 activator gene from one such plasmid in Saccharopolyspora erythraea drastically reduced expression from the cognate actI promoter, showing that host factors are required for optimal production of the activator protein itself. However, a low copy number expression vector pWIZ1 for the polyketide synthase DEBS1-TE, in which the promoter for the activator gene was replaced by the strong heterologous ermE* promoter of S. erythraea directed highly efficient production of polyketide synthase protein in Streptomyces cinnamonensis; and the levels of triketide lactone product found were up to 100-fold greater than were produced by the same plasmid in which actII-ORF4 was expressed from its own promoter. Ensuring appropriate expression of a specific activator protein should enable more convenient and consistent heterologous expression of genes in a broad range of actinomycete hosts.     

Repetitive extragenic palindromic sequences: a major component of the bacterial genome

We describe a remarkably conserved nucleotide sequence, the many copies of which may occupy up to 1% of the genomes of E. coli and S. typhimurium. This sequence, the REP (repetitive extragenic palindromic) sequence, is about 35 nucleotides long, includes an inverted repeat, and can occur singly or in multiple adjacent copies. A possible role for the REP sequences in regulation of gene expression has been thoroughly investigated. While the REP sequences do not appear to modulate differential gene expression within an operon, they can affect the expression of both upstream and downstream genes to a small extent, probably by affecting the rate of mRNA degradation. Possible roles for the REP sequence in mRNA degradation, chromosome structure, and recombination are discussed.     

Intergeneric transfer of the Enterococcus faecalis plasmid pIP501 to Escherichia coli and Streptomyces lividans and sequence analysis of its tra region

The nucleotide sequence of the transfer (tra) region of the multiresistance broad-host-range Inc18 plasmid pIP501 was completed. The 8629-bp DNA sequence encodes 10 open reading frames (orf), 9 of them are possibly involved in pIP501 conjugative transfer. The putative pIP501 tra gene products show highest similarity to the respective ORFs of the conjugative Enterococcus faecalis plasmids pRE25 and pAMbeta1, and the Streptococcus pyogenes plasmid pSM19035, respectively. ORF7 and ORF10 encode putative homologues of type IV secretion systems involved in transport of effector molecules from pathogens to host cells and in conjugative plasmid transfer in Gram-negative (G-) bacteria. pIP501 mobilized non-selftransmissible plasmids such as pMV158 between different E. faecalis strains and from E. faecalis to Bacillus subtilis. Evidence for the very broad-host-range of pIP501 was obtained by intergeneric conjugative transfer of pIP501 to a multicellular Gram-positive (G+) bacterium, Streptomyces lividans, and to G- Escherichia coli. We proved for the first time pIP501 replication, expression of its antibiotic resistance genes as well as functionality of the pIP501 tra genes in S. lividans and E. coli.