Study Of Low Molecular Active Factor Produced By Fungi

Two innovatory conclusions were got by biochemistry and bioinformatics study: i. Production of low molecular peptides by fungi is a universal phenomenon. ii. Two low molecular substance with antibacterial function were screened out, and it is hopeful they be used as novel antibiotics.1. Production of low molecular peptides by fungi is a universal phenomenon and there were synergic effects of the peptides and celiulases during native cellulose degradation.Function of HO · produced by low molecular peptides of fungi during cellulose degradation was reported for times. But all conclusions were based on studies of pure cellulose substrates. Whether this kind of peptide could degrade native cellulose as pure cellulose and whether it was ubiquitous in fungi had already been key factors to settle the question how to transform cellulose by microorganisms to use cellulose mass effectively and economically and how to develop resource and environment continually and harmonize. Fifty-seven cellulolytic fungi strains belonging to 47 species of White-rot, Brown-rot and Soft-rot fungi were tested, the results verified that the capacity of low molecular peptides for producing HO is a universal phenomenon in cellulolytic fungi. The synergic effects of the peptides and celiulases during native cellulose degradation were also found out. low molecular peptides produced by fungi were tested. Results showed that the peptides which could complex Fe³⁺ and reduce it to Fe²⁺ were universally in fungi. Furthermore, these abilities related to cellulose degradation. Typical cellulolytic fungi could complex Fe³⁺ strongly. These abilities were foundation to oxidation mechanism of HO · , because Fe²⁺ was necessary to Fenton reaction. TBA (Thiobarburic acid) method was used to test the activity of HO · produced by lowmolecular peptides which were generated by 57 cellulolytic fungi. Positive reactions of TBA were found in all of the low molecular components (MM<5KD) of excellular filter of 57 cellulolytic fungi cultured on cellulose media, which mean that capacity of low molecular peptides for producing HO ? was a universal phenomenon in cellulolytic fungi.The ability to reduce mucosity of carboxymethyl cellulose-Na (CMC-Na) related to HO ? producing were found by testing low molecular peptides of seven fungi. This indicated that HO ? can break the glycosidic bonds of CMC and then reduce mucosity of CMC-Na.Synergistic effect of low molecular peptides and cellulase from Trichoderima pseudokoningii S38, Penicillium sp., Alternaria alternata, Phytophthora sp., Ophiobolus graminis was studied and dry wheat straw, corn straw, and wood powder were used as substrates. The production of reducing sugars was selected as an index for efficiency of cellulose degradation. Tthe yield of reducing sugar was increased through the synergistic effects of low molecular peptides and cellulase from T. pseudokoningii S38, Penicillium sp., A. alternata on cellulose especially when the substrate was wood powder. While Phytophthora sp. and O. graminis were not typical cellulolytic fungi and their ability of HO production was very poor, so the yield of reduction sugar was not increase by synergistic effect. All results verified the function of SFGF on native cellulose degradation.Cellulose degradation could be promoted by oxygen because it involved oxidation and de-oxidation reaction.2. Antibiotic effects of HO ? depend on react condition(external experiment).It was a universal phenomenon that low molecular peptides generated by cellulolytic fungi could produce HO ? which could react with almost all kinds of organism. So we deduced that HO ? produced by cellulolytic fungi can anti-microbes generally. Antibiotic effects of HO ? generated by low molecular peptides were discussed. Results showed that HO ? could inhibit growth of Escherichia coli, but the effect was hard to measure because of the complex reaction system.Cellulolytic fungi with high ability of HO ? producing were selected to study antibacterial function of HO ? after reacting with Fe3+. And number of bacteria colonies was used as an index of antibacterial ratio.Fe3+could inhibit E. coli when its concentration was more than 10"5 mol/L. But HO ? produced by low molecular peptides showed its antibacterial function when concentration of Fe3+ was reduced. It was necessary to harmonize concentration of Fe3+ between HO ? production and non-inhibition to bacteria.It is sure that HO ? produced by low molecular peptides could inhibit bacteria. But because of the complex reaction system and multi-effects to bacteria, it is difficult to measure the inhibition ratio. When the inhibition of HO ? to A.-phage was tested, things went the same way.Effects of HO ? to native cellulose material and during the culture process of cellulolytic fungi had been verified, but its antibacterial function depended on react condition. The difference lie on the fact that E. coli was affected by Fenton reaction system. The above study we had done based on the unselective character of HO ? which result in non-resistance of pathogen. But now it is immature to use HO ? as an antibacterial directly.3. Two low molecular substance with antibacterial function were screened out from Penicillium sp.M03. Scientific study had been carried out and it was hopeful they be used as novel antibiotics to control resistant -bacteria.3.1 It was difficult for medicine selection because of the serious resistant of antibiotics. It was urgent to founding out novel antibiotics to control resistant pathogens. An low molecular weight (MW<5KD) antibiotic (AF) was screened out from fermentation filter of many fungi. The origin fungi was identified as Penicillium sp.M03 accooding to its morphology characteristic. Molecular range of AF was 1000-2000, and pi of AF was 3.6. AF was stable to 3 -lactamase, hydroxylamine, acid and alkali.Also, AF could keep activity for a long time at room temperature, and its activity could not reduce heated at 50°C for 30 min. AF could keep at least 50% activityboiling for 30 min. But melt after freezing reduced activity of AF. The above results manifested AF was not Penicillin.3.2 MIC and MBC of AF were 8 mg/ml and 32 mg/ml respectively measured by Double-dilution in test tubes. MIC to multi-resistant Staphylococcus aureu and to wild strain was similarly. Bacteria was not apt to resistant to AF, because MIC to passage S. aureu was not change when passage number was 200 in LB liquid medium with AF (concentration < MIC). AF could inhibit Gram-positive bacterium and Gram-negative bacterium, but not fungi. Seven in eight tested E.coli could not be inhibited by AF, while only a defective mutant could be. This maybe relate to the unique antibacterial mechanism of AF. AF showed inhibition to pathogen bacteria of human and plant, such as Sta.aureu, Streptococcus pneumoniae, Xanthomonas sp. and Erwinia sp.. All of these verified the study significance of AF.Growth curve of S.aureu in AF(lmg/ml) changed to reversed "V", which mean that AF not only kill bacteria but also caused bacterial cell self-melted or caused cytoplasm leakage and result in the reducing of medium turbidity. Scan electronic microscopy showed cell membrane of S.aureu had been damaged by AF, which result in leakage of cytoplasm, and so sunk surface of bacteria appeared. On the other hand, mucous layer around bacteria was hurt and its adhesive power reduced. Transmission electronic microscopy showed that inner-structure of S. aureu was hurt by AF^Nucleolus area disappeared and cytoplasm become confusion. From SEM and TEM it could be seen that there were relationship between hurt of inner-structure and of cell membrane. Antibacterial mechanism of AF related to damage of cell membrane and cytoplasm leakage. Moreover, AF could protect plants from being infected by pathogens.3.3 Variety of culture medium could be used by Penicillium sp.M03 to produce AF, bran/corn straw powder medium was the best. Growing on solid medium, Penicillium sp.M03 could suppress growth of bacteria before its colony. Some quantity of biomass of Penicillium sp.M03 was necessary for the produce of AF, but to much biomass was disadvantage for AF producing. NaNO3 was a profitable nitrogen source for AF producing. Maltose was the favorable carbon source whenfermented at 25 °C, while glucose was the favorable carbon source when fermented at 30°C. The optimum fermentation condition of AF was as the following: glucose 15g, K2HPO4 1 g, KC1 0.5g, NaNO3 3 g, MgSO4 -7H2O 0.5 g, FeSO4 0.01 g, distilled water 1L, pH 7 .0, fermentation at 30°C for5d.Antibacterial activity of AF increased with the decrease of temperature, especially at the temperature range 14°C³0°C. Low pH value enhanced the abilities of antibacterial, and AF lost all activity at pH 10.3.4 Purified by Sephadex LH-20 molecular sieve chromatography and HPLC, two antibacterial pure components AFl and AF2 were got. Their absorption spectrums were similar. They both had one peak at 200 ran. But absorption of AF2 was lower about ten times than AFl. Analysis showed that AF2 contained two saccharoses residue—glucose and xylose. It was unknown which monosaccharose AFl contained. Amino acid analysis showed that AFl contained no amino acid after acidolysis while it contained one rare amino acid. It means amino acid can be destructed by acidolysis. Analysis showed AF2 contained five amino acids: Ala, Glu, Gly, Asp and He. Many reported antibiotic peptides and glycopeptides contains rare amino acid which make normal amino acid analysis difficult.Structures of AFl and AF2 were studied through FT-IR, NMR and MALDI-TOF/MS. The results showed: AFl was consisted of a serial of similar aminoglycoside compounds whose molecular weight range was 627-1465. AF2 was glycopeptide. Its molecular weight was 1017 and consisted of fiver amino acids and a disaccharide.