β-聚糖酶的诱导合成及玉米芯半纤维素酶水解物木糖醇发酵酵母菌的筛选
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摘要
以富含木聚糖的植物纤维为原料，经酶水解和发酵是生产木糖醇的重要途径之一。本文以绿色木霉(Trichoderma viride)LH-011为产酶菌株，以经适当预处理的玉米芯为产酶碳源和诱导物，对β-聚糖酶的诱导合成的影响因素进行了系统研究。论文还对玉米芯半纤维素的酶水解及利用酶水解物直接发酵产生木糖醇的酵母菌进行了初步筛选。
β-聚糖酶诱导合成研究表明：碳源种类、浓度，氮源及碳氮比，初始pH值和培养温度等因素对β-聚糖酶合成都有一定的影响。以经适当预处理的玉米芯为主要碳源和诱导物，碳源浓度15g／L(以绝干料中碳元素含量计)，利用硫酸铵+尿素+蛋白胨为复合氮源，当控制碳氮比为6.4，初始pH值为4.8时，通过变温调控培养96小时(前24小时32±1℃，后期28±1℃)，纤维素酶活力(以CMC酶活计)和木聚糖酶活力分别达到了3.76U／ml和13.95U／ml。
利用自制β-聚糖酶对玉米芯粗木聚糖进行酶水解，当底物浓度25g／L，pH值4.8左右，酶用量为10％(v／v)，50℃恒温水浴，80r／min水解12小时以后还原糖浓度达到15.24g／L并趋于恒定，水解物中葡萄糖占3.13％，木糖占为19.17％，木低聚糖占45.75％。
以商品木糖为底物，从收集到的7株木糖发酵酵母菌中选出热带假丝酵母(Candida tropicalis)AY91009作为出发菌株。CandidatropicalisAY 91009在含有50g／L木糖和10g／L酵母浸膏的筛选培养基中，28℃摇瓶培养72小时木糖醇浓度达到了28.7g／L，木糖消耗量98.1％，转化率达到了0.59g／g。利用玉米芯半纤维素酶水解物对Candida tropicalis AY 91009经8代驯化培养，结合木糖平板筛选到一株较好的木糖醇产生菌热带假丝酵母(Candida tropicalis)LH-081，并用其进行了250ml摇瓶发酵生产木糖醇的初步试验，当培养基中还原糖浓度为15.24g／L，装料量为100ml，初始pH值5.4，温度28±1℃培养72小时(转速前24小时160r／min，后48小时100r／min)，木糖醇浓度为2.19g／L，还原糖利用率为28.7％，转化率0.50g／g。
论文研究结果表明，以预处理玉米芯诱导合成β-聚糖水解酶，用β-聚糖水解酶降解玉米芯半纤维素，利用玉米芯半纤维素酶水解物直接发酵制备木糖醇，路线上是行得通的，如果结合分离水解物中的未转化低聚糖，或者制备成富含木糖醇的木低聚糖类功能性食品，将能获得高附加值产品，使得整个过程获得较高的经济利益和社会效益。这为半纤维素的生物转化利用开辟了一条新的途径，同时也为今后发酵工艺参数的优化、水解酶的制备、木糖醇高产菌种的选育以及后续研究同步糖化与发酵工艺提供了参考。
Enzymatical hydrolysis of xylan-rich lignocellulosics followed by fermentation is one of the most important paths to produce xylitol. With pretreated com cob as carbon source and inducer, impacting factors of β-glycanase production by Trichoderma viride LH-011 were systematically studied in this thesis. Moreover, enzymatical hydrolysis of corn cob hemicellulose and screening of xylitol-producing yeast for fermentation of the hydrolysate to xylitol were primarily explored.
The experimental results show that the kind and concerntration of carbon source, the kind of nitrogen source and the C/N , the initial pH value and the culture temperature all have some effects on the biosynthesis of β -glycanase. When 15g/L pretreated com cob used as the main corbon source and inducer, mixed (NHU^SQj and urea and peptone as nitrogen source,regulating C/N 6. 4, initial pH value 4. 8, volume charge 50mL in 250 mL flask, 150 r/min, 32±1 ℃ the first 24 hours and 28±1℃ the rear, the CMC-ase activity and the xylanase activity respectively reached 3.76 U/ml and 13.95 U/ml after 96 hours cultivation.
The corn-cob hemicellulose hydrolysation has been done under the following conditions: the substrate concerntration was 25g/L, the pH value 4.8, self-made β -glycanases dosage 10% (v/v), 50± water bath,80r/min. As a result, the reducing sugars concentration reached 15.24 g/L after 12 hours and almost kept constantly, and glucose accouting for 3.13%, xylose 19.17% and xylo-oligomers 45.75% o
Candida tropicalis AY 91009 was selected by commercial xylose culture medium from the seven xylose-fermenting yeast strains bought from Institute of Microbilogy of Chinese Academy of Science and China Center for Type Culture Collection, which was then adapted to the corn-cob hemicellulose enzymatic hydrolysate (CCHEH) .Through eight-generation gradient CCHEH medium culturing, Candida tropicalis LH-081, a good xylitol-producing strain ,was screened and isolated by high concerntration xylose plate,, Primary study on bioconverting CCHEH into xylitol was carried out using Candida tropicalis LH-081 by 250ml flask batch fermentation process.The best results, a xylitol yield of 0.50g/g reducing sugar with an reducing sugar utilization efficiency of 28.7% , were achieved under the following conditions: reducing sugars concentration of CCHEH 15.24g/L, loading volume 100ml, nitrogen source 1.5g/L yeast extract and 1.5g/L peptone, initial pH value 5.4, inoculum size 10%(volume ratio), the first 24 hours, 160 r/min and
the later 48 hours, 100r/min , culture temperature 28±1 ℃ .
The result show that bioconversion the CCHEH into xylitol is not a very efficient but a very promising way. in order to realize the simultaneous saccharification and fermentation of the corn-cob hemicellulose for xylitol production, some more things must be done.
β-聚糖酶诱导合成研究表明：碳源种类、浓度，氮源及碳氮比，初始pH值和培养温度等因素对β-聚糖酶合成都有一定的影响。以经适当预处理的玉米芯为主要碳源和诱导物，碳源浓度15g／L(以绝干料中碳元素含量计)，利用硫酸铵+尿素+蛋白胨为复合氮源，当控制碳氮比为6.4，初始pH值为4.8时，通过变温调控培养96小时(前24小时32±1℃，后期28±1℃)，纤维素酶活力(以CMC酶活计)和木聚糖酶活力分别达到了3.76U／ml和13.95U／ml。
利用自制β-聚糖酶对玉米芯粗木聚糖进行酶水解，当底物浓度25g／L，pH值4.8左右，酶用量为10％(v／v)，50℃恒温水浴，80r／min水解12小时以后还原糖浓度达到15.24g／L并趋于恒定，水解物中葡萄糖占3.13％，木糖占为19.17％，木低聚糖占45.75％。
以商品木糖为底物，从收集到的7株木糖发酵酵母菌中选出热带假丝酵母(Candida tropicalis)AY91009作为出发菌株。CandidatropicalisAY 91009在含有50g／L木糖和10g／L酵母浸膏的筛选培养基中，28℃摇瓶培养72小时木糖醇浓度达到了28.7g／L，木糖消耗量98.1％，转化率达到了0.59g／g。利用玉米芯半纤维素酶水解物对Candida tropicalis AY 91009经8代驯化培养，结合木糖平板筛选到一株较好的木糖醇产生菌热带假丝酵母(Candida tropicalis)LH-081，并用其进行了250ml摇瓶发酵生产木糖醇的初步试验，当培养基中还原糖浓度为15.24g／L，装料量为100ml，初始pH值5.4，温度28±1℃培养72小时(转速前24小时160r／min，后48小时100r／min)，木糖醇浓度为2.19g／L，还原糖利用率为28.7％，转化率0.50g／g。
论文研究结果表明，以预处理玉米芯诱导合成β-聚糖水解酶，用β-聚糖水解酶降解玉米芯半纤维素，利用玉米芯半纤维素酶水解物直接发酵制备木糖醇，路线上是行得通的，如果结合分离水解物中的未转化低聚糖，或者制备成富含木糖醇的木低聚糖类功能性食品，将能获得高附加值产品，使得整个过程获得较高的经济利益和社会效益。这为半纤维素的生物转化利用开辟了一条新的途径，同时也为今后发酵工艺参数的优化、水解酶的制备、木糖醇高产菌种的选育以及后续研究同步糖化与发酵工艺提供了参考。
Enzymatical hydrolysis of xylan-rich lignocellulosics followed by fermentation is one of the most important paths to produce xylitol. With pretreated com cob as carbon source and inducer, impacting factors of β-glycanase production by Trichoderma viride LH-011 were systematically studied in this thesis. Moreover, enzymatical hydrolysis of corn cob hemicellulose and screening of xylitol-producing yeast for fermentation of the hydrolysate to xylitol were primarily explored.
The experimental results show that the kind and concerntration of carbon source, the kind of nitrogen source and the C/N , the initial pH value and the culture temperature all have some effects on the biosynthesis of β -glycanase. When 15g/L pretreated com cob used as the main corbon source and inducer, mixed (NHU^SQj and urea and peptone as nitrogen source,regulating C/N 6. 4, initial pH value 4. 8, volume charge 50mL in 250 mL flask, 150 r/min, 32±1 ℃ the first 24 hours and 28±1℃ the rear, the CMC-ase activity and the xylanase activity respectively reached 3.76 U/ml and 13.95 U/ml after 96 hours cultivation.
The corn-cob hemicellulose hydrolysation has been done under the following conditions: the substrate concerntration was 25g/L, the pH value 4.8, self-made β -glycanases dosage 10% (v/v), 50± water bath,80r/min. As a result, the reducing sugars concentration reached 15.24 g/L after 12 hours and almost kept constantly, and glucose accouting for 3.13%, xylose 19.17% and xylo-oligomers 45.75% o
Candida tropicalis AY 91009 was selected by commercial xylose culture medium from the seven xylose-fermenting yeast strains bought from Institute of Microbilogy of Chinese Academy of Science and China Center for Type Culture Collection, which was then adapted to the corn-cob hemicellulose enzymatic hydrolysate (CCHEH) .Through eight-generation gradient CCHEH medium culturing, Candida tropicalis LH-081, a good xylitol-producing strain ,was screened and isolated by high concerntration xylose plate,, Primary study on bioconverting CCHEH into xylitol was carried out using Candida tropicalis LH-081 by 250ml flask batch fermentation process.The best results, a xylitol yield of 0.50g/g reducing sugar with an reducing sugar utilization efficiency of 28.7% , were achieved under the following conditions: reducing sugars concentration of CCHEH 15.24g/L, loading volume 100ml, nitrogen source 1.5g/L yeast extract and 1.5g/L peptone, initial pH value 5.4, inoculum size 10%(volume ratio), the first 24 hours, 160 r/min and
the later 48 hours, 100r/min , culture temperature 28±1 ℃ .
The result show that bioconversion the CCHEH into xylitol is not a very efficient but a very promising way. in order to realize the simultaneous saccharification and fermentation of the corn-cob hemicellulose for xylitol production, some more things must be done.
引文
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