海洋侧孢短芽孢杆菌Lh-1抗菌活性物质的分离及特性研究

Purification and properties of an antimicrobial substance from marine Brevibacillus laterosporus Lh-1

摘要:

采用超滤、DEAE-Sephadex A25和CM-Sepharose FF离子交换层析及HPLC反相层析等步骤得到了海洋侧孢短芽孢杆菌(Brevibacillus laterosporus)Lh-1的抗菌活性物质,命名为R-1。HPLC纯化出的抗菌物质经质谱测定其分子量为1608.023Da,BIO-RAD等电聚焦测得其pI为8.55,氨基酸分析表明该物质由Leu、Tyr、Val、Ile、Lys、Gly、Met、Ser、Ala9种氨基酸组成。该抗菌物质具有极强的耐热、耐酸碱特性,在pH11.0~12.0条件下,121℃处理1h,其活力保持在75%以上。经3种蛋白酶(碱性蛋白酶、胰酶、胃蛋白酶)处理后,活性保持在80%以上。茚三酮反应阳性。推测R-1可能是低分子量的脂肽。抑菌试验表明对食品腐败菌、致病性革兰氏阴性、阳性菌及少数真菌均有抑菌活性。

Abstract:

An antimicrobial substance produced by Brevibacillus laterosporus isolated from the sea sediment was purified and characterized. The antimicrobial substance was purified by ultrafiltration，DEAE-Sepharose Fast flow chromatography，CM-Sepharose Fast flow chromatography and HPLC reversed phase column chromatography，and after the final purification step，one active fraction was obtained，designated R-1. The molecular weight (MW) was accurately determined by MALDI-TOF-MS as 1608.023 Da. And its pI was determined with Rotofor Cell BIO-RAD to be 8.55. Amino acid analysis of the purified R-1 showed that it was composed of Leu，Tyr，Val，Ile，Lys，Gly，Met，Ser and Ala. Most of them were hydrophobic and neutral amino acid except Lys which was a basic amino acid. And this accorded with pI of R-1. R-1 remained active over a wide temperature range and it also was active over a broad pH rang. R-1 was insusceptible to pancreatin，pepsin and alkaline proteinase. Agar radial diffusion assay showed that R-1 had low minimun bactericidal concentration against Gram-Positive Bacteria such as Streptococcus mutans，Staphylococcus aureaus，Clostridium and Gram- Negative Bacteria such as Escherichia coli，Pseudomonas putrefaciens. And R-1 had antibacterial activities against Candida albicans.