摘要：采用Plackett-Burman法考察了环境因素对产酸克雷伯氏菌降解氯氰菊酯的影响,并筛选出影响其降解效果的主要因素,即pH、温度和接种量。在此基础上,利用Box-Behnken实验设计和响应面分析法对其降解条件进行了优化。验证结果显示,当培养基中氯氰菊酯含量为200mg/L、pH为6.8、接种量(种子液OD6001.000)为5.8%(v/v)、250mL锥形瓶装液量为30mL、33℃培养120h时,产酸克雷伯氏菌对氯氰菊酯的降解率达到84.67%。

摘要：根据产酸克雷伯氏菌多聚半乳糖醛酸酶基因设计并筛选出一对实时荧光PCR特异性引物及Taq Man探针,建立了婴儿配方奶粉中产酸克雷伯氏菌实时荧光PCR检测方法。特异性实验结果表明,6株产酸克雷伯氏菌参考菌株出现了特异性扩增,18株近源参考菌株没有特异性扩增;灵敏度实验结果表明,婴儿配方奶粉中产酸克雷伯氏菌检测低限可到达10^(0)cfu/100 g数量级。该方法特异性好,检测灵敏度高,检测流程短,可在实际工作推广。

摘要：Ethanol is the main byproduct of anaerobic H2-producing fermentation in Klebsiella oxytoca HP1. Two moles of NAD(P)H are consumed to yield one mole of ethanol that may decrease bacterial hydrogen production. In this article the adhE gene that codes for acetaldehyde dehydrogenase was disrupted for the first time. A homologous recombination vector pTA-Str was constructed in which the adhE gene was disrupted by inserting an aminoglycoside-3'-adenyltransferase (aadA) gene. As expected, the vector includes the insertion 5′-adhE-aadA-adhE-3′. The amplified DNA fragment 5′-adhE-aadA-adhE-3′ from pTA-Str was transformed into K. oxytoca HP1 and one recombinant was obtained. PCR analysis of the resulting genomic DNA indicated the appropriate deletion and insertion. Compared with the H2-production of wild type K. oxytoca HP1, the hydrogen yield of the mutant increased by 16.07% and ethanol concentration decreased by 77.47%, suggesting that inactivation of the adhE gene in K. oxy- toca HP1 is a potential method for enhancing bacterial H2-production.

摘要：为了进一步了解2,3-丁二醇合成路径中关键酶的表达量与产量之间的关系,本研究根据Gen Bank中乙酰乳酸合成酶基因(bud B)的基因序列设计引物,以产酸克雷伯氏菌(Klebsiella oxytoca)HD79基因组DNA为模板,通过PCR扩增技术得到目标酶基因,目的片段全长1680 bp。以表达载体p RSFDuet-1为骨架,利用基因工程手段构建整合表达载体p R1SW-bud B。电转化法将其转化至菌株HD79感受态细胞中,通过高浓度Kan筛选和PCR双重鉴定验证后测定ALS酶活力。结果表明,乙酰乳酸合成酶基因整合表达载体构建成功,酶活可达1.0627 U/mg,比原始菌株提高了4.35倍。在一定程度上为实现2,3-丁二醇的工业化生产奠定基础。

摘要：乙酸是2,3-丁二醇典型代谢途径中的主要副产物之一,它的大量生成能够造成环境p H值的改变并且抑制菌体的生长,并对2,3-丁二醇的代谢产生影响。乙酸激酶是乙酸生成的关键酶,敲除其相关基因,理论上可以减少乃至消除乙酸的产生,从而提高2,3-丁二醇的产量和得率。本研究根据Klebisella oxytoca KCTC1686和Klebisella oxytoca HD79乙酸激酶基因ack序列的相似性设计引物,以产酸克雷伯氏菌基因组DNA和质粒载体p T-ack为模板,利用PCR克隆得到了乙酸激酶部分基因ack,长度为856bp,无碱基缺失。ORF分析发现该部分基因没有完整的开放阅读框,但是全部位于Klebisella oxytoca KCTC1686 ack基因的开放阅读框内。结果表明该片段为Klebisella oxytoca HD79乙酸激酶部分基因序列,为后续构建产酸克雷伯氏菌乙酸激酶突变株奠定了基础。