Increasing the Thermostability of Luciferase from Antarctic Krill by Rational Design for Biotechnological Applications  
[PERSON]  
1 Laboratory of Enzyme Engineering, Yellow Sea Fisheries Research Institute, Qingdao 266072, China; [EMAIL_ADDRESS] (Y.  
stable presence of ATP in living cells [3; 4; 5]. In the field of bacterial detection, the luminescence generated by the luciferase-catalyzed reaction is employed to quantify ATP released from bacterial cell lysates, thereby enabling the rapid and indirect enumeration of bacterial cells. For instance, [PERSON] et al. successfully utilized luciferase to detect contamination by _Listeria monocytogenesin_ food samples [6]. Similarly, [PERSON] et al. demonstrated the rapid and accurate detection of industrial pollutants in water environments using luminescent bacteria [7]. These studies highlight the versatility of luminescence-based methods in various detection applications. The development of genetically engineered bacteria has further expanded the potential of luminescence-based detection. By introducing luciferase genes into recipient bacterial strains through genetic engineering, the modified bacterial cells acquire the capacity to catalyze luminescence reactions [8]. In practical applications, these genetically engineered bacteria are introduced into the test system. Upon stimulation by specific target microorganisms, the engineered bacteria undergo bioluminescence reactions, producing detectable luminescence. The intensity of this luminescence is directly proportional to the concentration of the target microorganisms, thereby allowing for quantitative and directional safety testing. This approach offers a highly sensitive and specific method for detecting and quantifying specific microorganisms in complex environmental or biological samples. Moreover, the high sensitivity of luciferase-based imaging allows for the direct monitoring and tracking of tumor cell metastasis and growth by detecting the bioluminescent signals from cells labeled with the luciferase gene [9; 10; 11]. A notable example is the work of [PERSON] in 2006, who successfully monitored the interaction between the hepatitis C virus and host cells using a dual cis-trans luciferase virus reporter genome [12]. This innovative approach not only provided an intuitive means of observing the impact of drug factors on the virus but also served as a platform for evaluating the efficacy of viral inhibitors [13]. Luciferaseases are broadly categorized into bacterial and firefly luciferase [14; 15; 16]. A common limitation of most naturally occurring luciferase is their poor thermal stability. For instance, the North American firefly luciferase, a well-known enzyme involved in bioluminescence, becomes inactive after being stored at 37 \({}^{\circ}\)C for just 3 min in vitro [17; 18; 16]. This thermal instability poses a significant challenge for the application of luciferase in various experimental and clinical settings. Antarctic krill, a keystone species in the Antarctic ecosystem, is found at densities of 10,000-30,000 individuals per cubic meter and represents one of the most successful animal species on Earth in terms of biomass energy, with an estimated total of approximately 500 million tons. These organisms possess specialized biological fluorescent organs capable of producing fluorescence. These organs are strategically distributed across different parts of the krill's body: one pair in the eye stalks; another pair along the second to seventh thoracic and foot positions; and one in the abdomen. When threatened, Antarctic krill release an enzyme (luciferase) and a small molecular substrate (luciferin), which react in the presence of oxygen to emit bright violet fluorescence. This bioluminescent response not only masks the krill's shadow, rendering it \"invisible\" to predators, but also plays a crucial role in mating and nighttime aggregation behaviors. Our research team has made significant strides in this field by completing the whole-genome sequencing of Antarctic krill [19].