Su Caixin, Wang Dongmei, Yao Liming, Yu Zengliang
Ion Beam Bioengineering Key Lab, Institute of Plasma Physics, Chinese Academy of Sciences, 230031, Hefei 1126#, People's Republic of China.
J Agric Food Chem. 2006 Jun 14;54(12):4208-14. doi: 10.1021/jf0600556.
For the production of oligosaccharides from chitosan, a chitosanase-producing bacterium, S65, was isolated from soil. On the basis of phylogenetic analysis of the 16S rDNA gene sequence and phenotypic analysis, S65 was identified as a Bacillus sp. strain. This bacterium constitutively produced chitosanase in a culture medium without chitosan as an inducer. S65 chitosanase was homogeneously purified by DEAE Sepharose fast flow anion exchange followed by Superdex 75 size exclusion, and the molecular weight was 45 kDa according to SDS-PAGE. Enzyme analysis showed that the optimum pH and temperature of S65 were 6.0 and 65 degrees C, respectively. Catalytic activity was stable from pH 5.5-6.5 at temperatures below 40 degrees C, and the pI of chitosanase was about 6.0 as determined by a test tube method. S65 chitosanase degraded carboxymethyl cellulose (CMC) at the degree of about 5.3% relative to the value of soluble chitosan, but it cannot hydrolyze colloidal chitin and crystalline cellulose. Gene encoding was cloned and sequenced. The deduced amino acid sequence of the S65 exhibited the highest homology to those of family 8 glycanase, suggesting that the enzyme belonged to family 8.
为了从壳聚糖生产寡糖，从土壤中分离出了一株产壳聚糖酶的细菌S65。基于16S rDNA基因序列的系统发育分析和表型分析，S65被鉴定为芽孢杆菌属菌株。该细菌在没有壳聚糖作为诱导剂的培养基中组成性地产生壳聚糖酶。通过DEAE Sepharose快速流动阴离子交换，随后进行Superdex 75尺寸排阻，对S65壳聚糖酶进行了均一纯化，根据SDS-PAGE分析，其分子量为45 kDa。酶分析表明，S65的最适pH值和温度分别为6.0和65℃。在40℃以下的温度下，催化活性在pH 5.5-6.5范围内稳定，通过试管法测定，壳聚糖酶的pI约为6.0。S65壳聚糖酶相对于可溶性壳聚糖的值，以约5.3%的程度降解羧甲基纤维素（CMC），但它不能水解胶体几丁质和结晶纤维素。对编码基因进行了克隆和测序。S65推导的氨基酸序列与8族聚糖酶的氨基酸序列具有最高的同源性，表明该酶属于8族。
