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1、蜂巢形棉布载体固定化米根霉产果胶酶的半连续化发酵研究摘 要天然纤维素（如橘皮、烟梗）是一种可再生生物质资源，但由于纤维素、半纤维、木质素和果胶等交织在一起形成紧密复杂的结构，造成微生物利用天然纤维素生产乳酸、乙醇等的困难。米根霉在一定培养条件下能产生降解果胶质的酶系。果胶酶被广泛用于食品、纺织、生物技术等领域，其市场需求量日益增长。本文采用一种研究较少但有产果胶酶能力且富有商业价值的安全菌种米根霉，结合棉布载体固定化细胞的技术进行发酵产果胶酶。采用DNS法测定果胶酶（PEC）酶活力和用粘度法测定聚半乳糖醛酸内切酶（Endo-PG）酶活力。首先从纯果胶发酵产果胶酶出发，考察了固定化发酵与游离发酵
2、差别，优化了纯果胶培养基，优化了培养条件并进行半连续发酵试验，研究了发酵液部分果胶酶酶学性质；然后优化烟梗浸液培养基，优化了培养条件并进行半连续试验，还研究了发酵液中纤维素酶活力变化情况。首先，对纯果胶发酵产果胶酶进行研究。在优化前培养基和培养条件下，固定化和游离发酵相比，固定化发酵48h就达到最大产酶量，缩短发酵时间24h，提高产酶量115%。采用单因素和正交试验法优化培养基，结果表明影响产果胶酶的因素依次为：果胶Tween80Zn2+硫酸铵。发酵培养基为：果胶2.5%，硫酸铵1.5%，ZnSO40.6 mmol/L，Tween80 0.15% ，K2HPO4 0.4%，KH2PO4 0.4
3、%；在此基础上采用单因素法优化培养条件，结果为：果胶2.5%，硫酸铵1.5%，ZnSO40.6 mmol/L，Tween80 0.15% ，K2HPO4 0.4%，KH2PO4 0.4%，转速190r/min、装液量50ml/250ml、发酵温度30C、发酵初始pH 5.0、初始孢子浓度0.75106个/mL，培24h，PEC酶活力和Endo-PG酶活力分别为973.47U/mL和165.08U/mL。通过对粗酶液作用pH和温度的研究，得到PEC和Endo-PG为酸性果胶酶，最适pH为4.5，PEC和Endo-PG最适温度分别为45 C和55 C。半连续发酵试验结果表明，在第5批次出现最高PE
4、C酶活力和Endo-PG酶活力，分别为1253.95U/mL和181.94U/mL，分别比首次提高29.8%和18.3%。然后，对烟梗浸液发酵产果胶酶进行研究。采用单因素和正交试验法优化培养基，结果表明影响产果胶酶的因素依次为：烟梗硫酸铵Zn2+Tween80。发酵培养基为：烟梗10%，硫酸铵1.5%，ZnSO40.6 mmol/L，Tween80 0.05%，K2HPO4 0.2%，KH2PO4 0.2%。在此基础上采用单因素法优化培养条件，结果为：烟梗10%，硫酸铵1.5%，ZnSO40.6 mmol/L，Tween80 0.05%，K2HPO4 0.2%，KH2PO4 0.2%，发酵初始
5、pH 5.0，初始孢子浓度0.5106，30C，装液量50mL，转速170r/min，培养48h，PEC酶活力和Endo-PG酶活力最高分别为361.27U/mL和49.22U/mL。烟梗浸液发酵96h，出现纤维素酶活力高峰，滤纸酶活力和羧甲基纤维素钠酶活力分别为33.25U/mL和83.13U/mL。半连续发酵试验结果表明，在第2批次出现最高PEC酶活力和Endo-PG酶活力，分别为430.83U/mL和51.73U/mL，PEC酶活力比首批次提高约22.8%。关键词：米根霉，果胶酶，聚半乳糖醛酸内切酶，固定化，半连续化发酵ABSTRCTNatural cellulose is one of
6、 the renewable resources, such as orange peel and tobacco stem, which are made of cellulose, half fiber, lignin and pectin. The intertwined combination among them leads to form a tight and complex structure, which is hard for microorganisms to digest. Hence, the production of lactic acid or ethanol
7、through using natural cellulose by microorganisms is faced with problems. Rhizopus oryzae has the ability to produce pectinolytic enzymes to degrade pectin.The remove of pectin can make separation of fiber, fiber and lignin easier. There is a growing market demand of pectinolytic enzymes for the fac
8、t that pectinolytic enzymes are widely used in the filed of food, textile, pharmaceutical, paper making, biological technology and so on. Rhizopus oryzae, which owns the ability to degrade pectin and is rich in comercial value, is used to produce pectinolytic enzymes in this research. The immobilize
9、d Rhizopus oryzae with matrix composed of asterisk-configuration fibrous matrices in a honeycomb-shaped, the production of pectinolytic enzymes were carried out from citrus pectin and tobacco stem pectin. Pectinolytic enzymes (PEC) activity was determined by DNS assay and endopolygalacturonase (Endo
10、-PG) activity was measured by viscosity method. Fistly, the pure pectin was selected as the sbustrate for fermentation. Then, the coparison of enzymes production between immobilized cells and free cells was made. After that, optimization of culture medium and growth conditions was investigated throu
11、gh single factor variable analysis and so on. Also, some properties of the enzyme were under reasearch. Finally, optimization of culture medium made from tobacco stem was investigated through single factor variable analysis and orthogonal experiment. Optimization of growth conditions was investigate
12、d through single factor variable analysis. Six batches of semi-continuous fermentation were carried out in shake flasks.First of all, the study of pure pectin was carried out. Immobilized cells had a higher production of PEC over free cells. Immobilized cells had a max production in 48 hours, which
13、saved 24 hours compared with free cells and improved production by 115%. Results of single factor and orthogonal experimental with immobilized showed the order of factors influencing the production of pectinase was: pectin Tween80 ZnSO4(NH4)2SO4.The suitable culture media was composed of pectin2.5%,
14、 (NH4)2SO4 1.5%, ZnSO40.6 mmol/L ,Tween80 0.15%, K2HPO4 0.4%，KH2PO4 0.4%，rotation speed 190 r/min, liquid volume 50 ml/250 ml, temperature 30C, initial pH 5.0, initial spore concentration 0.75106 /mL. After 24 hours, the PEC activity and Endo-PG activity were 973.47U/mL and 165.08U/mL separately. Ef
15、fects of pH and temperature on crude enzymes fluid were carried out.PEC and Endo-PG might be acidic pectinolytic enzymes. The optimum pH was 4.5 for PEC and Endo-PG, optimum temperature was 45Cand 55C respectively. The highest PEC activity and Endo-PG activity were 1253.95U/mL and 181.94U/mL in the
16、5th batch, about 29.8% and 18.3% higher than the 1st batch. Secondly, optimization of culture medium made from tobacco stem was investigated through single factor variable analysis and orthogonal experiment. Results showed the order of factors influencing the production of pectinase was: tobacco stem (NH4)2SO4ZnSO4Tween80. The suitable culture media was composed of tobacco stem 10%, (NH4)2SO4 1.5%, ZnSO40.6 mmol/L,Tween80 0.05%, K2HPO4 0.2%
