Shri AMM Murugappa Chettiar Research Centre, Taramani, Chennai, 600113, India.
Appl Biochem Biotechnol. 2011 Jul;164(5):666-80. doi: 10.1007/s12010-011-9166-6. Epub 2011 Jan 28.
Pink pigmented facultative methylotrophs (PPFM) isolated from water samples of Cooum and Adyar rivers in Chennai and soil samples of forests located in various districts of Tamil Nadu, India were screened for cellulase production using carboxymethylcellulose agar (CMC agar) medium. The strains showed wide variations in the production of clearing zones around the colonies on CMC agar medium flooded with Congo red. CMCase and filter paper assays were used to quantitatively measure the cellulase activity of 13 PPFM strains. Among the strains, Methylobacterium gregans, MNW 60, MHW 109, MSF 34, and MSF 40 showed cellulolytic activity ranging from 0.73 to 1.16 U mL(-1) with wide temperature (35-65°C) and pH (5 to 8) tolerance. SDS-PAGE analysis of the crude enzyme of PPFM strain MNW 60 exhibited several protein bands, and zymogram analysis revealed two dimeric cellulase bands with molecular mass of ~92 and 42 kDa. Scanning electron microscopic studies revealed significant morphological differences between the cells grown in normal and CMC amended medium. The strain MNW 60 was identified as Methylobacterium sp. based on biochemical, physiological, and morphological analyses, and the methylotrophic nature was authenticated by the presence of mxaF gene, encoding methanol dehydrogenase as a key indicator enzyme of methylotrophs, with 99% similarity to Methylobacterium lusitanum. With the 16S ribosomal RNA sequence showing 97% similarity to M. lusitanum strain MP2, this can be proposed as a novel taxon of the genus Methylobacterium. The study forms the first detailed report on the extracellular cellulase production by pink pigmented Methylobacterium sp., and it is expected that this might be the basis for further studies on cellulase production by PPFMs to explore the molecular mechanism, strain improvement, and large-scale cellulase production for its application.
从印度泰米尔纳德邦不同地区森林的土壤样本以及钦奈的 Cooum 和 Adyar 河流的水样中分离出粉红色可培养需甲基营养型（PPFM），并用羧甲基纤维素琼脂（CMC 琼脂）培养基筛选产纤维素酶。这些菌株在刚果红饱和的 CMC 琼脂培养基上产生的菌落周围的透明圈大小差异很大。用 CMCase 和滤纸分析法定量测量 13 株 PPFM 菌株的纤维素酶活性。在这些菌株中，Methylobacterium gregans、MNW 60、MHW 109、MSF 34 和 MSF 40 表现出 0.73 至 1.16 U mL-1 的纤维素酶活性，具有较宽的温度（35-65°C）和 pH（5-8）耐受性。PPFM 菌株 MNW 60 的粗酶 SDS-PAGE 分析显示出几个蛋白条带，而同工酶分析显示出两个二聚体纤维素酶条带，分子量约为 92 和 42 kDa。扫描电子显微镜研究表明，在正常和添加 CMC 的培养基中生长的细胞之间存在显著的形态差异。根据生化、生理和形态分析，菌株 MNW 60 被鉴定为 Methylobacterium sp.，并且存在编码甲醇脱氢酶的 mxaF 基因，作为甲基营养型的关键指示酶，这证实了其甲基营养型，与 Methylobacterium lusitanum 具有 99%的相似性。16S 核糖体 RNA 序列与 M. lusitanum MP2 菌株的相似度为 97%，因此可以将其作为 Methylobacterium 属的一个新分类群。这项研究首次详细报道了粉红色 Methylobacterium sp. 的细胞外纤维素酶的产生，预计这可能为进一步研究 PPFMs 的纤维素酶生产提供基础，以探索分子机制、菌株改良和大规模纤维素酶生产及其应用。
