一株耐盐纤维素酶海洋曲霉的筛选及产酶条件研究

Screening of a Cellulase Producing Halotolerant Strain Marine Aspergillu sp and Its Fermentation Conditions

从东海海底泥中分离到了一株耐盐和高产内切葡聚糖酶(CMCase)及β-葡萄糖苷酶(β-Gluase)的海洋曲霉ZJUBE-1,根据形态特征及18SrDNA分析,初步鉴定该菌为黑曲霉(Asper-gillus niger)。通过液体发酵产酶优化,获得最佳培养基组成,质量分数分别为:麸皮2.5%,氯化铵(或蛋白胨、磷酸二氢铵)1%,含NaCl 3.0%～3.6%的人工海水作无机盐液。发酵条件为100mL三角瓶装培养液30mL,在起始pH 6.0、37℃,接种量为6%,180r/min条件下振荡培养7d,测得发酵液中CMCase、β-Gluase酶活及滤纸酶活(FPA)分别为5.3、5.4、0.75U/mL。该菌在NaCl质量分数24%下仍具产酶能力,所产3种纤维素酶的最适反应温度均为65℃,最适反应pH分别为4.0、5.0、5.0;在NaCl质量分数为2%～12%(β-Gluase在2%～16%)范围内,3种酶活力维持较高水平。该菌及其所产纤维素酶的高度耐盐性预示着其在废水处理、纺织业、海洋养殖、海产品加工或海藻的能源化利用等高盐环境下可能有较好的应用前景。

In this manuscript,a halotolerant marine strain Aspergillus sp ZJUBE-1 possessed the production of endoglucanase and β-Gluase with high cellulase activities was isolated from the submarine mud sample of Zhejiang inshore,and identified as Aspergillus niger by two different methods of both the morphology and 18S rDNA gene sequences.The optimal medium compositions for liquid-state fermentation for cellulase production by strain ZJUBE-1 were determined and listed as follows: wheat bran 2.5%,NH4Cl or(NH4)H2PO4 1%,NaCl 3~ 3.6%.Under the condition of shaking culture for 7 days,at 37 ℃,the initial medium pH 6.0 and rotating speed 180 r/min,the maximum activities of carboxymethyl cellulase activity(CMCase),β-glucosidases(β-Gluase) and filter paper activity(FPA) were respectively reached at 5.3 U/mL-1,5.4 U/mL-1 and 0.75 U/mL,respectively.Moreover,the result showed that the strain still has cellulase productive ability,even the NaCl concentration exceed 24%.The studies on enzymatic properties showed that the optimal reaction temperature for CMCase,β-Gluase and FPA was the same temperature at 65 ℃,but the optimal pH value of three enzyme production were different,corresponding to 4.0,5.0,5.0,respectively.It was also found that even on the condition of 2~12% NaCl(β-Gluase is about in 2~16%),the production of three cellulase keep higher activities.These above-mentioned results indicated that the strain ZJUBE-1 has an extensive applications under high salinity environment in many fields,such as wastewater treatment,textile industry,HYPERLINK 'http://dict.cnki.net/dict_result.aspx?searchword=%e6%b5%b7%e4%ba%a7%e5%93%81%e5%8a%a0%e5%b7%a5&tjType=sentence&style=&t=marine+products+processing' marine products processing and the utility of seaweed for energy source for its high salt-tolerant property in the near future.