纤维素酶产生菌的筛选及功能菌NF-101的全基因分析与产酶工艺优化  

Screening of Cellulase-producing Bacteria,Whole-gene Analysis,and Optimization of the Enzyme Production Process for the Functional Bacterium NF-101

作　　者：张智[1] 王蒙爱 冯丽荣 郑莹 盛新和 章圣龙 蒋士龙 解庆刚 ZHANG Zhi;WANG Mengai;FENG Lirong;ZHENG Ying;SHENG Xinhe;ZHANG Shenglong;JIANG Shilong;XIE Qinggang(Heilongjiang Provincial Key Laboratory of Forest Food Resources Utilization,School of Forestry,Northeast Forestry University,Harbin 150040,China;Heilongjiang Guohong Energy Conservation and Environmental Protection Co.Ltd.,Harbin 150040,China;Heilongjiang Feihe Dairy Co.Ltd.,Harbin 150040,China)

机构地区：[1]东北林业大学林学院黑龙江省森林食品资源利用重点实验室,黑龙江哈尔滨150040 [2]黑龙江国宏节能环保有限公司,黑龙江哈尔150040 [3]黑龙江飞鹤乳业有限公司,黑龙江哈尔滨150040

出　　处：《现代食品科技》2024年第11期107-118,共12页Modern Food Science and Technology

基　　金：黑龙江省“揭榜挂帅”科技攻关项目(2021ZXJ03B01);黑龙江省“百千万”工程科技重大专项(2020ZX07B01-3-2)。

摘　　要：我国北方地区是主要的农作物产区,以种植秸秆为主。为了合理利用农作物的自然资源,并寻找高效纤维素降解菌,该研究从东北三省20个地点采集100件样品。经初筛分离得到可分解纤维素的菌种2052株,初步鉴定为细菌1207株、霉菌598株、及放线菌195株,保存至-80℃超低温冰箱,以作为建立降解纤维素酶功能菌库储备菌种。经复筛得到16株产纤维素酶能力较高的功能菌,经16S rDNA及生理生化鉴定,为粘质沙雷氏菌、地衣芽孢杆菌、耐盐芽孢杆菌等,其中产酶能力最强的为地衣芽孢杆菌(Bacillus licheniformus)NF-101。经单因素及响应面优化,NF-101最佳产酶培养基为羧甲基纤维素钠0.75 wt.%,蛋白胨0.6 wt.%,磷酸二氢钾0.2 wt.%,硫酸镁0.05 wt.%。最佳工艺条件为接种量4%,初始pH值7.0,温度37℃,培养时间72 h,优化后滤纸酶活达到181.22 U/mL,与优化前相比提高了约3.63倍。对NF-101进行全基因分析,证明该菌为高效降解纤维素酶优势菌株,并为纤维素酶降解实验提供优势菌株及实验依据。Northern China is a major agricultural region,primarily producing cereal crops,which are associated with large amounts of residual straw.To efficiently utilize this natural agricultural resource and identify efficient cellulose-degrading bacteria,100 samples were collected from 20 sites in three northeastern provinces.After preliminary screening, 2 052 strains capable of decomposing cellulose were isolated, which were provisionally identified as 1 207 strains of bacteria, 598 strains of mold, and 195 strains of actinomycetes. These strains were stored in an ultra-low temperature freezer at -80 ℃ for subsequent establishment of a functional cellulase-degrading strain library. After re-screening, 16 functional bacteria with high cellulase production ability were obtained, which were identified based on 16S rDNA analysis and physiological and biochemical identification as Serratia marcescens, Bacillus licheniformis, and halotolerant Bacillus, among which B. licheniformis NF-101 was found to be characterized by the most efficient enzyme production. Following single-factor and response surface optimization, the optimal enzyme-producing medium for cultivating NF-101 was identified as a medium comprising sodium carboxymethylcellulose 0.75 wt.%, peptone 0.6 wt.%, potassium dihydrogen phosphate 0.2 wt.%, and magnesium sulfate 0.05 wt.%. The optimal process conditions were a 4% inoculation amount, initial pH of 7.0, temperature of 37 ℃ , and incubation time of 72 h. Optimized filter enzyme activity reached 181.22 U/mL, which represented an approximate 3.63-fold increase compared with that prior to optimization. Whole- gene analysis of NF-101 proved that it was a dominant strain for efficient cellulase-mediated degradation. It also provided information on a dominant strain and an experimental basis for cellulase degradation experiments.

关 键 词：菌种选育 纤维素酶 产酶条件优化 全基因分析 

分 类 号：TQ925[轻工技术与工程—发酵工程]