高产纤维素酶里氏木霉菌株诱变选育及其发酵条件优化
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摘要
试验旨在研究高产纤维素酶里氏木霉诱变选育与发酵条件优化。采用常压室温等离子体(ARTP)诱变法处理里氏木霉,获得产纤维素酶高的突变菌,并对其产酶发酵条件进行优化。通过单因素实验研究发酵时间、硫酸铵浓度、微晶纤维素浓度、接种量及搅拌速度等对里氏木霉产酶的影响。在单因素的基础上,通过正交实验对里氏木霉产酶的工艺参数进行优化。结果表明,在诱变时间240 s条件下筛选到1株突变里氏木霉ATR-4,其滤纸酶活(FPU)最高可达2.01 U·mL~(-1)。对突变里氏木霉菌株ATR-4的发酵条件优化,筛选得到最佳产酶培养条件为:发酵时间78 h,硫酸铵浓度1 g·L~(-1),接种量10%,搅拌速度400 r·min~(-1)。在此条件下进行验证实验,最高酶活可达4.57 U·mL~(-1)。本研究结果表明,常压室温等离子体(ARTP)诱变可有效对里氏木霉进行诱变育种,改善其产酶能力。
This experiment was conducted to study the mutagenesis breeding and optimal fermentation condition optimization of high yield cellulase enzyme of Trichoderma reesei. Mutants of Trichoderma reesei induced by atmospheric and room temperature plasma(ARTP) were screened for cellulase production. The fermentation conditions of cellulase enzyme production were studied. Five factors including fermentation time, ammonium sulfate concentration, microcrystalline cellulose concentration, inoculation amounts and stirring speed were studied for their effect on the yield of cellulase enzyme. On the base of the single factor experiments, the optimal fermentation conditions for the cellulase production were obtained by orthogonal experiments. The results showed that the optimum parameters for cellulase production were: fermentation time 78 h, ammonium sulfate concentration 1 g·L~(-1),inoculation amounts 10%, stirring speed 400 r·min~(-1). The cellulase activity was 4.57 U·mL~(-1). This study showed that the mutagenesis of atmospheric and room temperature plasma(ARTP) was effective in mutagenesis breeding of Trichoderma reesei, thus to improve enzyme producing ability.
This experiment was conducted to study the mutagenesis breeding and optimal fermentation condition optimization of high yield cellulase enzyme of Trichoderma reesei. Mutants of Trichoderma reesei induced by atmospheric and room temperature plasma(ARTP) were screened for cellulase production. The fermentation conditions of cellulase enzyme production were studied. Five factors including fermentation time, ammonium sulfate concentration, microcrystalline cellulose concentration, inoculation amounts and stirring speed were studied for their effect on the yield of cellulase enzyme. On the base of the single factor experiments, the optimal fermentation conditions for the cellulase production were obtained by orthogonal experiments. The results showed that the optimum parameters for cellulase production were: fermentation time 78 h, ammonium sulfate concentration 1 g·L~(-1),inoculation amounts 10%, stirring speed 400 r·min~(-1). The cellulase activity was 4.57 U·mL~(-1). This study showed that the mutagenesis of atmospheric and room temperature plasma(ARTP) was effective in mutagenesis breeding of Trichoderma reesei, thus to improve enzyme producing ability.
引文
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[2] Trivedi N, Gupta V, Kumar M, et al. An alkali-halotolerant cellulase from Bacillus flexus isolated from green seaweed Ulva lactuca[J]. Carbohydrate Polymers, 2011, 83(2):891-897.
[3]黄晓梅,赵红晓,范金霞,等.一株高产纤维素酶绿色木霉菌株诱变选育与发酵研究[J].东北农业大学学报, 2018, 49(6):22-31.
[4]韩峰,孙彩云.拟康氏木霉(Trichoderma pseudokoningii)UVⅢ产纤维素酶液体发酵研究[J].工业微生物, 2002(1):28-30.
[5]王加友,赵彭年,杨德玉,等.一株纤维素分解菌的筛选、鉴定及其对玉米秸秆的降解效果[J].生物技术进展, 2018, 8(2):132-139.
[6]李豪,白光剑,兰楠,等.高产纤维素酶菌株的筛选、鉴定及产酶特性研究[J].中国畜牧兽医, 2019, 46(3):711-718.
[7]孟庆山.人工锌指蛋白介导调控的里氏木霉纤维素酶生产[J].生物工程学报, 2019, 35(1):81-90.
[8] Saloheimo M, Pakula T M. The cargo and the transport system:secreted proteins and protein secretion in Trichoderma reesei(Hypocrea jecorina)[J]. Microbiology, 2012, 158:46-57.
[9] Li Y, Liu C, Bai F, et al. Overproduction of cellulase by Trichoderma reesei RUT C30 through batch-feeding of synthesized low-cost sugar mixture[J]. Bioresource Technology, 2016, 216:503-510.
[2] Trivedi N, Gupta V, Kumar M, et al. An alkali-halotolerant cellulase from Bacillus flexus isolated from green seaweed Ulva lactuca[J]. Carbohydrate Polymers, 2011, 83(2):891-897.
[3]黄晓梅,赵红晓,范金霞,等.一株高产纤维素酶绿色木霉菌株诱变选育与发酵研究[J].东北农业大学学报, 2018, 49(6):22-31.
[4]韩峰,孙彩云.拟康氏木霉(Trichoderma pseudokoningii)UVⅢ产纤维素酶液体发酵研究[J].工业微生物, 2002(1):28-30.
[5]王加友,赵彭年,杨德玉,等.一株纤维素分解菌的筛选、鉴定及其对玉米秸秆的降解效果[J].生物技术进展, 2018, 8(2):132-139.
[6]李豪,白光剑,兰楠,等.高产纤维素酶菌株的筛选、鉴定及产酶特性研究[J].中国畜牧兽医, 2019, 46(3):711-718.
[7]孟庆山.人工锌指蛋白介导调控的里氏木霉纤维素酶生产[J].生物工程学报, 2019, 35(1):81-90.
[8] Saloheimo M, Pakula T M. The cargo and the transport system:secreted proteins and protein secretion in Trichoderma reesei(Hypocrea jecorina)[J]. Microbiology, 2012, 158:46-57.
[9] Li Y, Liu C, Bai F, et al. Overproduction of cellulase by Trichoderma reesei RUT C30 through batch-feeding of synthesized low-cost sugar mixture[J]. Bioresource Technology, 2016, 216:503-510.