Cloning, characterization, and molecular application of a beta-agarase gene from Vibrio sp strain V134

V134, a marine isolate of the Vibrio genus, was found to produce a new beta-agarase of the GH16 family. The relevant agarase gene agaV was cloned from V134 and conditionally expressed in Escherichia coli. Enzyme activity analysis revealed that the optimum temperature and pH for the purified recombinant agarase were around 40 degrees C and 7.0. AgaV was demonstrated to be useful in two aspects: first, as an agarolytic enzyme, the purified recombinant AgaV could be employed in the recovery of DNA from agarose gels; second, as a secretion protein, AgaV was explored at the genetic level and used as a reporter in the construction of a secretion signal trap which proved to be a simple and efficient molecular tool for the selection of genes encoding secretion proteins from both gram-positive and gram-negative bacteria.

Cloning, characterization, and molecular application of a beta-agarase gene from Vibrio sp strain V134

V134, a marine isolate of the Vibrio genus, was found to produce a new beta-agarase of the GH16 family. The relevant agarase gene agaV was cloned from V134 and conditionally expressed in Escherichia coli. Enzyme activity analysis revealed that the optimum temperature and pH for the purified recombinant agarase were around 40 degrees C and 7.0. AgaV was demonstrated to be useful in two aspects: first, as an agarolytic enzyme, the purified recombinant AgaV could be employed in the recovery of DNA from agarose gels; second, as a secretion protein, AgaV was explored at the genetic level and used as a reporter in the construction of a secretion signal trap which proved to be a simple and efficient molecular tool for the selection of genes encoding secretion proteins from both gram-positive and gram-negative bacteria.

Nisin高产菌株选育及应用的研究

本论文以牛奶为原料分离出42株球菌，经鉴定其中15株为乳链球菌（Streptococcus lactis）。对15株乳链球菌进行Nisin效价测定后，确定SN-21为诱变的出发菌株（598.1IU/ml,NO.95培养基），经过硫酸二乙酯和紫外线的多次诱变后，选育出一株Nisin高产菌株（1514IU/ml,CM培养基），命名为S. L. 21 (Streptococcus lactis 21)。通过对S. L. 21发酵条件的选优，其Nisin效价达到1862IU/ml。采用紫外吸收光谱法确定S. L. 21发酵产物中的抑菌物质为Nisin。在Nisin高产菌株选育的同时，开创了Nisin应用于蕨菜罐头的加工贮藏研究。通过在蕨菜罐头中添加0.1g/kg的Nisin，降低了蕨菜罐头的杀菌温度（100 ℃）和杀菌时间（15min），保证了蕨菜的品质，提高了蕨菜罐头的贮藏安全性，这为低酸易软烂野生蔬菜食品的加工贮藏提供了一条重要途径。100 ℃，15min杀菌条件下，在加工的蕨罐头中，未添加Nisin的处理和添加苯甲酸钾（1.0g/kg）的处理，均出现胀罐，爆罐现象。经分析确定蕨菜罐头胀罐原因是由污染的细菌产气引起的。通过分离污染菌优势类群得到8株芽孢杆菌，经系统的细菌学鉴定，4~#菌株为巨大芽孢杆菌（Bacillus megaterium），8~#菌株为短小芽孢杆菌（Bacillus pumilus），其余为枯草芽孢杆菌（Bacillus subtilis）。经进一步产气试验证明5~#菌株产气快，产气量大，是引起蕨菜罐头胀罐的污染优势种，经研究发现蕨菜罐头污染菌优势种是一种枯草芽孢杆菌的新变种，命名为枯草芽孢杆菌嗜热耐盐新变种（Bacillus sutilis n. var. sp.），这株新变种具有耐热（90 ℃），耐盐（10%NaCl），产生的特点。对蕨菜罐头污染菌优势种（5~#）进行防腐剂抑菌试验发现其对Nisin敏感（MIC 500ppm），而对苯甲酸钠（MIC 1.5%）和山梨酸钾（MIC 3%）不敏感，抑菌率试验进一步证明，在允许应用范围内，只有Nisin对蕨菜罐头防腐有良好的效果，而苯甲酸钠和山梨酸钾抑菌效果均不理想。在Nisin对污染菌优势种（5~#）的溶菌作用试验中，通过电镜可以观察到，Nisin的作用首先是破坏细胞壁，细胞膜，造成细胞内物质外流，严重溶菌结果，导致污染细菌死亡。以上关于Nisin应用于蕨菜罐头防腐，蕨菜罐头污染菌优势类群的分析，污染菌优势种的研究的试验结果均为国内外首次报导。总之，通过在蕨菜罐头中添加Nisin来抑制污染菌优势种的生长敏殖，进而达到降低杀菌强度的目的，对于保证蕨菜固有的品质及今后蕨菜等野生资源的开发具有重要的意义。