用基因组改组技术提高出芽短梗霉XYE-7的木聚糖酶活性的研究
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摘要
木聚糖是β-1，4-木糖的聚合物，是自然界中继纤维素之后的第二丰富的多糖。它占农业废料干重的20％-30％。它丰富的储量表明木聚糖酶将在生物转化，造纸制浆等方面有着巨大应用前景。
从广西大学农场果园中筛选到一株产木聚糖酶的酵母菌。经过形态学和分子生物学的鉴定，发现该菌属于Dothioraceae座囊菌纲科，Aureobasidium短梗霉属，其18SrDNA序列与AureobasidiumpullulansY68具有最高的相似性，初步命名为Aureobasidiumpullulans XYE-7。
经研究发现XYE-7合成木聚糖酶的最佳条件为：培养基由0.5％木聚糖、0.5％硫酸铵、0.2％酵母膏、0.1％磷酸二氢钾、0.95％硫酸镁组成，pH值2.0；发酵温度为35℃，培养时间为80h。
菌株XYE-7木糖诱导的木聚糖酶粗酶液的酶学性质为：该酶作用的最适温度是70℃，最适pH值为2.5；该酶在60℃以下时稳定性较好，高于60℃时酶活性下降变快；该酶在pH值2.0-5.0时有很好的稳定性，说明木糖诱导的XYE-7木聚糖酶在酸性范围内稳定。它的km值为10.1mg·mL~(-1)。
蔗渣木聚糖诱导的木聚糖酶的酶学性质为：该酶作用的最适温度是65℃，最适pH值为7.0；该酶在60℃以下时稳定性较好，60℃保温1h后仍然保留87％的活性，高于60℃时酶活性下降较快；该酶在pH值4.0-9.0时有很好的稳定性，说明XYE-7木聚糖酶有很宽的pH稳定范围。其km值为3.63mg·mL~(-1)。
XYE-7菌株制备原生质体的最佳条件为：采用20mL的酵母完全培养基培养菌体18h；预处理的巯基乙醇的浓度为0.25％，EDTA的浓度为0.05mol／L；预处理时间10min；酶液的高渗离子浓度为1.2mol／LMgSO_4；纤维素酶和蜗牛酶2％，酶解时间为1.5h。
紫外诱变得到酶活提高的菌株作为基因组改组的出发菌株，通过四轮的改组，得到18株酶活提高的菌株，其中一株菌的酶活比改组前出发菌株的酶活提高了50％，比野生型菌株的酶活提高了78％。
Xylan,a heteropolymer of (β-1-4)-linked xylose,is the next mostabundant natural polysaccharide after cellulose and accounts for 20to 30％dry weight of agricultural residues.Its abundance indicatesthat xylanolytic enzymes can play an important role inbioconversion,in the preparation of cellulose pulps,in fibre liberationtechnology, etc.
With the method of plate, a yeast-like strain which couldproduce extracellular xylanases, was isolated from soil. According toits morphological, physiological properties, it should belong toAureobasidium pullulans. The strain was named as Aureobasidiumpullulans XYE-7.
Through single factor tests, the optimal medium andfermentation conditions were determined, and it consisted 0.5％xylan as carbon source、0.5％(NH_4)SO_4and 0.2％barmene as nitrogen source、0.1％KH_2PO_4、initial pH 2.0 and culturetemperature 35℃for 80 h.
The strain can produce two kinds of different xylanases.Sugarcane xylan induced the strain to produce alkaline xylanase, andxylose can induce acid xylanase. According to the studies of theirenzymology properties, the xylanase induced by xylose is acidophilic.The pH optimum of acidophilic xylanase was 2.5 and thetemperature optimum was 70℃. The enzyme retained greater than80％of the original activity between pH 2.0 and 4.0. The thermalstability was determined at temperatures from 40 to 90℃for 30 min.The enzyme remained stable up to 60℃, but it lost the activity at70℃.
The xylanase induced by sugarcane xylan is basophilic. Theenzyme showed relatively high activity under basic conditions withan optimum of pH7.0 and stable at pH values between pH 4.0 and9.0.its temperature optimum was 65℃. The enzyme thermal stabilityis the same as the xylanase induced by xylaose. It remained stable upto 60℃, but it lost activity at70℃.
The conditions of protoplast preparation and regeneration fromthe XYE-7 were studied. Results showed that the 18-hour-oldmycelia,when subjected 2％celluanse and 2％helicase solution in1.2Mol/L MgSO4 (pH value 6.0)at 30℃for 1.5 hours with gentle sbaking, produced an eficient yield of protoplasts.
We have used genome shuffling to improve the xylanaseactivities of XYE-7, We have obtained several strains that activityhigher than the wide type strain by UV mutation.Eighteen strainswere obtained after the four rounds of shuffling. The hignest activityimproved half-fold than that of UV mutant.
从广西大学农场果园中筛选到一株产木聚糖酶的酵母菌。经过形态学和分子生物学的鉴定，发现该菌属于Dothioraceae座囊菌纲科，Aureobasidium短梗霉属，其18SrDNA序列与AureobasidiumpullulansY68具有最高的相似性，初步命名为Aureobasidiumpullulans XYE-7。
经研究发现XYE-7合成木聚糖酶的最佳条件为：培养基由0.5％木聚糖、0.5％硫酸铵、0.2％酵母膏、0.1％磷酸二氢钾、0.95％硫酸镁组成，pH值2.0；发酵温度为35℃，培养时间为80h。
菌株XYE-7木糖诱导的木聚糖酶粗酶液的酶学性质为：该酶作用的最适温度是70℃，最适pH值为2.5；该酶在60℃以下时稳定性较好，高于60℃时酶活性下降变快；该酶在pH值2.0-5.0时有很好的稳定性，说明木糖诱导的XYE-7木聚糖酶在酸性范围内稳定。它的km值为10.1mg·mL~(-1)。
蔗渣木聚糖诱导的木聚糖酶的酶学性质为：该酶作用的最适温度是65℃，最适pH值为7.0；该酶在60℃以下时稳定性较好，60℃保温1h后仍然保留87％的活性，高于60℃时酶活性下降较快；该酶在pH值4.0-9.0时有很好的稳定性，说明XYE-7木聚糖酶有很宽的pH稳定范围。其km值为3.63mg·mL~(-1)。
XYE-7菌株制备原生质体的最佳条件为：采用20mL的酵母完全培养基培养菌体18h；预处理的巯基乙醇的浓度为0.25％，EDTA的浓度为0.05mol／L；预处理时间10min；酶液的高渗离子浓度为1.2mol／LMgSO_4；纤维素酶和蜗牛酶2％，酶解时间为1.5h。
紫外诱变得到酶活提高的菌株作为基因组改组的出发菌株，通过四轮的改组，得到18株酶活提高的菌株，其中一株菌的酶活比改组前出发菌株的酶活提高了50％，比野生型菌株的酶活提高了78％。
Xylan,a heteropolymer of (β-1-4)-linked xylose,is the next mostabundant natural polysaccharide after cellulose and accounts for 20to 30％dry weight of agricultural residues.Its abundance indicatesthat xylanolytic enzymes can play an important role inbioconversion,in the preparation of cellulose pulps,in fibre liberationtechnology, etc.
With the method of plate, a yeast-like strain which couldproduce extracellular xylanases, was isolated from soil. According toits morphological, physiological properties, it should belong toAureobasidium pullulans. The strain was named as Aureobasidiumpullulans XYE-7.
Through single factor tests, the optimal medium andfermentation conditions were determined, and it consisted 0.5％xylan as carbon source、0.5％(NH_4)SO_4and 0.2％barmene as nitrogen source、0.1％KH_2PO_4、initial pH 2.0 and culturetemperature 35℃for 80 h.
The strain can produce two kinds of different xylanases.Sugarcane xylan induced the strain to produce alkaline xylanase, andxylose can induce acid xylanase. According to the studies of theirenzymology properties, the xylanase induced by xylose is acidophilic.The pH optimum of acidophilic xylanase was 2.5 and thetemperature optimum was 70℃. The enzyme retained greater than80％of the original activity between pH 2.0 and 4.0. The thermalstability was determined at temperatures from 40 to 90℃for 30 min.The enzyme remained stable up to 60℃, but it lost the activity at70℃.
The xylanase induced by sugarcane xylan is basophilic. Theenzyme showed relatively high activity under basic conditions withan optimum of pH7.0 and stable at pH values between pH 4.0 and9.0.its temperature optimum was 65℃. The enzyme thermal stabilityis the same as the xylanase induced by xylaose. It remained stable upto 60℃, but it lost activity at70℃.
The conditions of protoplast preparation and regeneration fromthe XYE-7 were studied. Results showed that the 18-hour-oldmycelia,when subjected 2％celluanse and 2％helicase solution in1.2Mol/L MgSO4 (pH value 6.0)at 30℃for 1.5 hours with gentle sbaking, produced an eficient yield of protoplasts.
We have used genome shuffling to improve the xylanaseactivities of XYE-7, We have obtained several strains that activityhigher than the wide type strain by UV mutation.Eighteen strainswere obtained after the four rounds of shuffling. The hignest activityimproved half-fold than that of UV mutant.
引文
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