篮子鱼内脏粗提液制备长心卡帕藻和细齿麒麟菜原生质体的初步研究

摘要

Protoplast of Kappaphycus and Eucheuma, two main traditional carrageenan-producing seaweeds, were prepared using crude enzyme extracts of Siganus fuscessens viscus from Sep, 2011 to Jan, 2013 at our tropical seaweed experimental station in Lingshui, Hainan. Both the stomach and liver of S. fuscessens viscus as well as the young branches of Kappaphycus and Eucheuma were pre-homogenized. Filtrate of S. fuscessens was used as the crude enzymatic extracts to digest the sendimented pellets of Kappaphycus and Eucheuma. Then, the total protoplast output and protoplast yield per gram of the algal homogenate pellets exposed to gradients of pH, temeprature and enzymic extracts were compared. The results showed that the protoplasts from Eucheuma were easily prepared compared to Kappaphycus, and that the amount of protoplast obtained depended on the digestion time, dosage of enzyme extract and the algal pellets. The more the algal homogenate pellets were added in the tested range (0.1-0.4 g fresh weight), the higher the total protoplast output and the lower protoplast yield per gram biomass were obtained. Meanwhile, the more enzyme extract was added, the higher the total protoplast output and the protoplast yield per gram biomass were harvested. Prolonging the enzymatic digestion time could linearly improve the total protoplast output and the protoplast yield per gram. The pH and temperature also significantly affected the protoplast production. The optimal pH and temperature for protoplast preparation were pH 6.0 and 25℃, respectively. Based on the above studies, a optmized mode for Kappaphycus and Eucheuma protoplast preparation was suggested as below:0.1 g homogenized algal pellets, 3 ml crude enzyme extract from 0.15 g homogenate of S. fuscessens stomach and liver with 50 mM phosphate buffer (pH 6.0), then adjusted the mixture pH to 6.0 and incubated at 25℃ for≥48 h.%于2011年9月至2013年1月，在海南陵水热带海藻实验基地，利用篮子鱼(Siganus fuscessens)内脏研磨制备消化酶，开展了酶解制备大型产胶红藻长心卡帕藻(Kappaphycus alvarezii)和细齿麒麟菜(Eucheuma denticulatum)的原生质体研究，观察了不同酶解条件对两种产胶海藻原生质体产量和产率的影响。结果表明，细齿麒麟菜原生质体得率高于长心卡帕藻；在藻泥与酶浓度配比中，长心卡帕藻或细齿麒麟菜藻泥越多，所制备出的原生质体产量越高，而其制备效率却越低；在温度(20~30℃)梯度中，有最高原生质体产量和效率的酶解温度为25℃；在pH 6.0~8.0的实验酶解梯度中，酶解pH 6.0时的酶解效果最好；在12~48 h实验酶解时间梯度中，酶解时间为48 h时原生质体产量和得率最高；酶解时间越长，长心卡帕藻或细齿麒麟菜原生质体制备效率越高。为此建议在25℃和 pH6.0条件下，适当增加海藻藻泥用量和延长酶解时间，以更有效获得原生质体。