Kinetic and thermodynamic investigation of Rhodanese synthesized by enhanced Klebsiella oxytoca JCM 1665 strain: a comparative between the free and immobilized enzyme entrapped in alginate beads.

IF 2 4区 生物学 Q3 BIOCHEMICAL RESEARCH METHODS
Babamotemi Oluwasola Itakorode, Dorcas Ibukunoluwa Itakorode, Nkem Torimiro, Raphael Emuebie Okonji
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引用次数: 0

Abstract

Klebsiella oxytoca JCM 1665 was subjected to extracellular rhodanese production using a submerged fermentation technique. The organism was further engineered for higher cyanide tolerance and rhodanese yield using ethylmethanesulfonate as a mutagen. Mutagenesis resulted in an improved mutant with high cyanide tolerance (100 mM) and rhodanese yield (26.7 ± 0.67 U/mL). This yield was 4.34-fold higher than the wild strain (6.15 ± 0.65 U/mL). At temperatures ranging from 30 to 80 °C, the first-order thermal denaturation constant (Kd) for free enzyme increases from 0.00818 to 0.0333 min-1 while the immobilized enzyme increases from 0.003 to 0.0204 min-1. The equivalent half-life reduces from 99 to 21 minutes and 231 to 35 minutes, respectively. Residual activity tests were used to assess the thermodynamic parameters for both enzyme preparations. For the free enzyme, the parameters obtained were enthalpy (29.40 to 29.06 kJ.mol-1), entropy (-194.24 to -197.50 J.mol-1K-1) and Gibbs free energy (90.20 to 98.80 kJ.mol-1). In addition, for immobilized rhodanese, we obtained enthalpy (40.40 to 40.07 kJ.mol-1), entropy (-164.21 to - 165.20 J.mol-1K-1) and Gibbs free energy (91.80 to 98.40 kJ.mol-1. Regarding its operational stability, the enzyme was able to maintain 63% of its activity after being used for five cycles. Immobilized K. oxytoca rhodanese showed a marked resistance to heat inactivation compared to free enzyme forms; making it of utmost significance in many biotechnological applications.

增强型氧合克雷伯氏菌 JCM 1665 菌株合成罗丹酶的动力学和热力学研究:游离酶与海藻酸珠子固定酶的比较。
利用浸没式发酵技术,对克雷伯氏菌(Klebsiella oxytoca)JCM 1665 进行了细胞外菱锰生产。利用甲基磺酸乙酯作为诱变剂,进一步改造了该菌株,使其具有更高的氰化物耐受性和铑产量。诱变产生的改良突变体具有较高的氰化物耐受性(100 mM)和铑产量(26.7 ± 0.67 U/mL)。该产量比野生菌株(6.15 ± 0.65 U/mL)高出 4.34 倍。在 30 至 80 °C 的温度范围内,游离酶的一阶热变性常数(Kd)从 0.00818 min-1 增加到 0.0333 min-1,而固定化酶则从 0.003 min-1 增加到 0.0204 min-1。等效半衰期分别从 99 分钟和 231 分钟缩短到 21 分钟和 35 分钟。残余活性测试用于评估两种酶制剂的热力学参数。对于游离酶,获得的参数为焓(29.40 至 29.06 kJ.mol-1)、熵(-194.24 至 -197.50 J.mol-1K-1)和吉布斯自由能(90.20 至 98.80 kJ.mol-1)。此外,对于固定化菱锰,我们得到了焓(40.40 至 40.07 kJ.mol-1)、熵(-164.21 至 -165.20 J.mol-1K-1)和吉布斯自由能(91.80 至 98.40 kJ.mol-1)。在操作稳定性方面,该酶在使用五个周期后仍能保持 63% 的活性。与游离酶形式相比,固定化的 K. oxytoca rhodanese 具有明显的抗热失活能力,因此在许多生物技术应用中具有极其重要的意义。
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来源期刊
Preparative Biochemistry & Biotechnology
Preparative Biochemistry & Biotechnology 工程技术-生化研究方法
CiteScore
4.90
自引率
3.40%
发文量
98
审稿时长
2 months
期刊介绍: Preparative Biochemistry & Biotechnology is an international forum for rapid dissemination of high quality research results dealing with all aspects of preparative techniques in biochemistry, biotechnology and other life science disciplines.
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