摘要
在木霉纤维素酶水解植物纤维过程中,添加外源β-葡萄糖苷酶是提高可发酵性单糖得率、降低酶用量的一种行之有效的方法。因此,外源β-葡萄糖苷酶的活力及其使用成本能否满足工业化应用的需要至关重要。
论文以制备高活力外源β-葡萄糖苷酶及多次重复利用β-葡萄糖苷酶为目标,优化了β-葡萄糖苷酶高产菌的培养条件并对产酶过程进行了pH值和补料调控;分离纯化了两种胞外β-葡萄糖苷酶酶和一种胞内β-葡萄糖苷酶酶,研究了其酶学特性;建立了超滤法、丙酮沉淀法、固定化酶法回收利用β-葡萄糖苷酶的方法及其工艺;并对β-葡萄糖苷酶的基因进行了克隆和表达。主要结果如下:
(1)筛选获得β-葡萄糖苷酶高产菌株A.niger-NL-1,第4 d时,酶活力最大值达到4.7 U/ml。在分泌β-葡萄糖苷酶的过程中,该菌株能产生较高活力的内切葡聚糖酶和外切葡聚糖酶,滤纸酶活达到0.62 IU/ml。并且,粗酶液中β-葡萄糖苷酶和内切葡聚糖酶具有较强的耐酒精特性。
(2)产酶时适宜的初始pH值为5.0。将产酶期的pH值控制在4.0左右有利于β-葡萄糖苷酶的分泌,120 h时酶活力达到6.12 U/ml。
(3)产酶过程中经过四次补加麸皮,酶活力提高了近3倍。补料发酵工艺中,适宜的麸皮初始浓度为3%,在补料总量一定时,采用递减补料方式效果最好,其酶活力达到7.0 U/ml。分批补料培养是一种很好的提高酶产量的方法。
(4)采用盐析、疏水层析、阴离子交换层析、凝胶过滤等步骤,纯化获得两种胞外β-葡萄糖苷酶,它们的单亚基分子量分别为114.6 KD和70.3 KD。高分子量的酶具有耐高糖特性,以对硝基苯酚-β-D-葡萄糖苷为底物,葡萄糖的抑制常数K_i为41.01 mmol/L。两种酶均不需要依赖金属离子维持其活性,能被一定浓度的乙醇、甲醇、正丁醇、乙酸乙酯激活。
(5)纯化获得一种胞内β-葡萄糖苷酶,其分子量大小为122.7 KD。甲醇、乙醇、正丁醇、丙酮和乙酸乙酯等有机溶剂对该酶具有显著的激活作用。添加30%甲醇、30%乙酸乙酯、40%正丁醇、40%丙酮时,它们的相对酶活力分别为153%、162%、184%和146%。该类酶在合成工业中将具有很好的应用前景。
(6)选择截流分子量30 KD超滤膜回收纤维二糖水解液中β-葡萄糖苷酶比较适宜。连续超滤回收不同批次的纤维二糖酶解液中β-葡萄糖苷酶,第一轮的酶回收率、平均膜通量分别为99.5%和109.4 L/m~2.h;第二十轮回收的β-葡萄糖苷酶为初始加酶量的90%以上,且平均膜通量为79.2 L/m~2.h。利用丙酮回收水解液中β-葡萄糖苷酶时,丙酮与水解液的体积比在0.75～1.0之间比较适宜。用-20℃丙酮沉淀2 h,酶回收率为95.7%。连续沉淀不同批次的纤维二糖酶解液时,第15次的酶回收率仍能在50%左右,可节省所需酶量的78.97%。
(7)以海藻酸钠为载体制备的固定化β-葡萄糖苷酶重复利用20次仍能保持90%以上的酶解得率。以进料速度为1.5 ml/min,1.0 ml/min连续酶解时,酶解得率分别达到96.65%和99%。与单独采用木霉纤维素酶相比,在β-葡萄糖苷酶总活力与滤纸酶活之比为0.5(FPA=2.0 IU/ml)的条件下,固定化β-葡萄糖苷酶与木霉纤维素酶协同作用降解滤纸纤维素和微晶纤维素60 h,它们的水解得率分别增加了20.4%和29.3%。
(8)克隆了片段大小为2582 bp的黑曲霉β-葡萄糖苷酶基因,构建了β-葡萄糖苷酶基因表达载体pPICZα-A-bgl1,通过转化成功获得了重组毕赤酵母。
(9)在适宜的条件下,重组酵母一步式发酵的第5d酶活力达到11.29 IU/ml,产酶能力明显高于二步式,且产酶周期大大缩短。利用二次电转化技术获得了多拷贝数的重组子,其表达量比一次电击获得的优良重组子增加了2.7倍,比出发菌株高出5倍多,实现了高效表达。
通过研究,本论文在四个方面取得了成果:(1)获得了β-葡萄糖苷酶的高产菌株,建立了产酶过程中pH调控和分批补料的方法与工艺。(2)酶的纯化及其性质的研究为获得更适合于植物纤维原料酶水解的β-葡萄糖苷酶或拓宽其新的应用领域打下了基础。(3)建立了超滤法、丙酮沉淀法、固定化酶法回收利用β-葡萄糖苷酶的方法及其工艺,为建立β-葡萄糖苷酶辅助降解纤维原料酶解液的新模式或新工艺提供了理论依据。(4)成功克隆出黑曲霉β-葡萄糖苷酶的基因,并在毕赤酵母中实现了高效表达,显示出了很好的工业化应用潜力。
In the process of hydrolysis of lignocellulosic material with cellulase produced by Trichoderma viride,supplying additionalβ-glucosidase was an effective way of increasing the yield of fermentable monosaccharide,decreasing the consumption and lowering the cost of enzyme.So the supplementaryβ-glucosidase activity and its cost are the significant factors for application in industries.
Studies in this dissertation were focused on the fermentation techniques for production ofβ-glucosidase at a high level by Aspergillus niger,purification and characterization the intracellular and extracellularβ-glucosidase from Aspergillus niger,recycleβ-glucosidase techniques of ultrafiltration,acetone precipitation and enzyme immobilization,and Clone the and expression gene ofβ-glucosidase.The main results are as follows:
(1)The highβ-glucosidase producing strain A.niger-n1-1 was selected from the experimental strains and its highest activity,up to 4.7 U/ml,was synthesized by A.niger-n1-1 at 96 h.Besidesβ-glucosidase,the higher exoglucanases and endoglucanases were synthesized by the strain during the same fermentation.The filter paper activity(FPA)reached 0.62 U/ml.
(2)The initial pH value of enzyme production was 5.0.Regulated pH value around 4.0 in the fermentation period the enzyme activity reached 6.12 U/ml at 120 h.and it would be favorable for the secretion ofβ-glucosidase.
(3)β-glucosidase activity increased by nearly 3 times after fourth batch feed bran during the fermentation.It worked the best when the initial concentration of bran was 3%and fed with descending mode.Under this conditions,the enzyme activity reached 7.0U/ml.the results showed that feed-batch culture was a favorable way to enhance the enzyme production.
(4)Two kinds of extracellularβ-glucosidase were purified to homogeneity from an Aspergillus niger by salting out,hydrophobic interaction chromatography,negative ion exchange chromatography,gel layer chromatography and other processes.Their single subunit molecular weight was 114.6 KD and 70.3 KD respectively.The one with higher molecular weight was highly glucose-tolerant.Its K_i was 41.01mmol/l using p-nitrophenyl-β-D-glucopyranoside as substrates.Metal ions had no effects on the two enzymes.It could be activated by a certain concentration of ethanol,methanol,1-butanol and acetoacetate.
(5)Obtained a kind of intracellularβ-glucosidase by purification and its molecular weight was 122.7 KD.It could be obviously activated by methanol,ethanol,1-butanol,acetone and acetoacetate.The relative enzyme activities were 153%,162%,184%,146%with additions of 30%methanol,30%acetoacetate,40%1-butanol,40%acetone.This kind of enzyme will have favorable applicative prospects in the synthetical industries.
(6)The 30 KD membrane was selected to recoverβ-glucosidase from cellobiose hydrolytic liquor when using ultrafiltration technique.In the signal batch,theβ-glucosidase recovery rate and the average permeation flux were 99.5%and 109.4%L/m~2.h respectively. The amount of recoveredβ-glucosidase from the 20~(th)batches of cellobiose hydrolytic liquor would still be maintained 90%of the initial total enzyme activity and the average permeation flux reached 79.2 L/m~2.h.When using organic solvent precipitation technique,it was suitable that the acetone and cellobiose hydrolytic liquor at the volume ratio of 0.75:1 to 1:1.The enzyme recovery rate was up to 95.7%by using -20℃acetone as precipitator for 2h.50% enzyme activity could still be maintained and 78.97%amount of required enzyme was saved when recycleβ-glucosidase 15 batches by acetone precipitation.
(7)β-glucosidase was immobilized by sodium alginate as carrier.The yield of enzymatic hydrolysis kept higher than 90%during 20 batches.When the rate were 1.5ml/min and 1.0ml/min,the yield reached 96.65%and 99%respectively.The synergetic hydrolysis processes on filter paper and Avicel were carrier out by the cellulase of T.viride and the immobilizedβ-glucosidase.On the condition of the ratio of totalβ-glucosidase activity to filter paper activity was 0.5 and FPA=2.0 IU/ml,at 60h,the degradation rate of filter paper and Avicel was increased by 20.4%and 29.3%respectively compared to that using T.viride only.
(8)Cloned theβ-glucosidase gene of A.niger that had 2582bp.Construct the gene expression vector pPICZα-A-bg11 ofβ-glucosidase and obtained the recombinant Pichia pastoris by transformation.
(9)Optimized the conditions to induce the recombinant Pichia pastoris to express.In the one-step method the activity reached 11.29U/ml on 5~(th)day which had high enzyme productivity than the two-step method and the enzyme yielding period was greatly shorten.Obtained the recons GS115 bg11-38 with relaxed copy by using 2~(th)electric transformation.It was showed that the expression amount of GS115 bg11-38 was increased by 2.7 times compared with the excellent recons GS115 bg11-3 got by 1~(th)electric transformation by shake-flask cultivation,5 times of that of the primary strain.So actualize the expression with high efficiency.
There are fruits in four aspects to summarize:(1)Obtained the highestβ-glucosidase producing strain from the 17 strains of Trichoderma,Basidiomycetes and Aspergillus. Established the techniques of pH regulation and feed-batch fermentation in the yielding process. (2)Provided a reference and direction for selecting more suitableβ-glucosidase for the enzyme hydrolysis of lignocellulosic resources and broadeningβ-glucosidase application by purification and characterization of three kind ofβ-glucosidase from A.niger.(3)Established the techniques of recycleβ-glucosidase using ultrafiltration,acetone precipitation and immobilized enzyme method.Provided theoretical basis for building new techniques or modes of hydrolyzing lignocellulosic material with an aid ofβ-glucosidase.(4)Cloned theβ-glucosidase gene of A. niger successfully and expressed in Pichia pastoris with high efficiency.So it showed great applicative potential in industries.
引文
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