Recovery Of Enzyme Activity Research Articles

Key role of streambed moisture and flash storms for microbial resistance and resilience to long‐term drought

Abstract Spatial and temporal widening of drought periods together with occurrence of flash storm events are consequences of global change affecting temporary stream ecosystems. Streambed heterotrophic microbes and the key biogeochemical processes they carry on could be endangered by the strengthening of drought episodes. Here, we performed a 165‐day experiment through 12 streambed sediment columns to study heterotrophic microbial functional and structural responses to long‐term drought. Two sediment depths (surface and hyporheic) and leaf litter were monitored under three treatments: control (maintained in wet, pool‐like conditions), dry (5 months of drought), and dry–storm (5 months of drought including two flash storms). All treatments were then followed by rewetting. Surface sediment followed by leaf litter was the most affected by the long‐term drought as shown by the reduction of polysaccharidic enzyme activities and litter decomposition rate, although lignin decomposition was not affected. This resulted in a greater use of recalcitrant compounds which might be due to reduced labile organic matter sources and the greater resistance of fungi than bacteria to drought. Moreover, in surface sediment, bacterial viability and algal biomass were reduced while the production of extracellular polymeric substances was intensified with dryness, suggesting its potential role as survival strategy. Hyporheic sediments appeared more resistant to long‐term drought, and this might be linked to its slightly higher water content (2.5%) than the surface (0.5%) during drying together with a greater content of fine material that allowed fungi and bacteria to survive and extracellular enzyme activities to be maintained. Microbial resilience to long‐term drought in sediment and leaf litter was promoted by the flash storms as shown by the fast recovery of enzyme activities, bacterial viability and leaf litter decomposition rate in the dry–storm treatment, once rewetted. Storms might liberate previously occluded carbon sources that fuel the fast microbial reactivation as well as providing sediment moisture for microbial survival. Our results highlight that long‐term drought may compromise stream biogeochemical processes linked to organic matter decomposition and that microbes are highly sensitive to minimal water content changes. Thus, long‐term drought consequences could be mitigated by occasional precipitations or by natural streambed moisture.

Use of α-amylase/silica particle suspensions to optimize cleaning in a simulated cleaning-in-place system

The activity and stability of α-amylase on silica particle suspensions were studied by analyzing the influence of time, temperature, enzyme concentration and silica particle concentration. The recovery of enzymatic activity was also explored using different centrifugation and reconstitution cycles and experimental times, comparing the results with those obtained without particles. Higher recovery of enzymatic activity was obtained by centrifugation at 60 °C and with a ratio particle concentration/enzyme concentration higher than 20. The cleaning of heat-treated starch retained in stainless steel was analyzed in a Cleaning-In-Place device (CIP) using enzymatic solutions with and without silica particles. The utilization of enzymatic solutions without microparticles enabled 36% of activity to be recovered by centrifugation. When silica particles were included in the washing solutions, the detergency recovered was up to 50%, this not being significantly affected by the storage time, nor the number of centrifugation and washing cycles.

Optimization of [CnPy]Cl (n=2,4,6) ionic liquid aqueous two-phase system extraction of papain using response surface methodology with box-behnken design

Abstract This work used ionic liquid aqueous two-phase (ILATP) extraction technology to obtain high-purity and high-activity papain. Papain was extracted using [CnPy]Cl(n=2,4,6)-K2HPO4 ILATP systems. Response surface methodology (RSM) was developed to optimize the conditions. Firstly, the effects of the reagent, temperature and pH on papain activity were investigated. Secondly, the influences of the salt concentration, ionic liquid concentration, papain dosage, pH and temperature on the partition behaviour of papain were investigated. The overall desirability (OD) of the enzyme activity recovery and partition coefficient was response indicator. Furthermore, the absorption spectrum of papain before and after extraction was characterized by UV–vis absorption spectroscopy. Finally, the purity of papain extracted by ILATP was analysed by SDS-PAGE electrophoresis. The order of the effect of various factors on papain extraction was: K2HPO4 concentration, [C4Py]Cl concentration, pH and papain dosage. A high temperature is not conducive to the extraction of papain from an ILATP system. The extraction efficiency of the [C4Py]Cl-K2HPO4 system is superior to that of the [C2Py]Cl and [C6Py]Cl systems. The maximum OD value 0.8985 was obtained under the conditions of 0.35 g/mL K2HPO4, 0.25 g/mL [C4Py]Cl, pH of 7.87, papain dosage of 2.17 mg/mL at 30℃. The UV spectra of papain before and after extraction were the same. The specific activity of papain extracted by ILATP was 4120.17 U/mg, which was 1.88-fold the purity of commercial papain. This work provides theoretical guidance for optimized extraction process parameters and the preparation of high-activity enzymes.

Overexpression of secreted sucrose isomerase in Yarrowia lipolytica and its application in isomaltulose production after immobilization

Isomaltulose production by bacterial fermentation was limited, due to generation of undesirable products and reduced yields. Isomaltulose production using sucrose isomerase (SIase) catalyzed methods was expected to be more applicable, but was hampered by low SIase activity and lack of a secreted SIase producer. Here, we aimed to obtain high levels of secreted SIase by overexpressing the SIase gene from Pantoea dispersa UQ68J in Yarrowia lipolytica, a successful host for efficient secretory expression, with a newly characterized strong constitutive promoter. After optimization of the culture medium, the engineered strain JD secreted SIase with an activity of 49.3 U/mL. The recombinant SIase was effectively immobilized onto polyvinyl alcohol-alginate, and the enzymatic activity recovery rate was up to 82.4%. The stability of the SIase was significantly improved by immobilization. Batch production of isomaltulose catalyzed by the immobilized SIase was performed under optimal conditions, generating 620.7 g/L isomaltulose with a yield of 0.96 g/g. The conversion rate of sucrose after 13 batches remained above 90%. These results demonstrated that the proposed SIase expression and immobilization method was promising in the industrial production of isomaltulose.

Assessment of CYP2D6 re-activation after inhibitory effect of MDMA using tramadol as a probe.

Abstract Background In recent years, the use of tramadol as a probe drug for human cytochrome p450 2D6 (CYP2D6) has been investigated. The objective of this study was to assess the recovery of rat CYP2D1 enzymatic activity after mechanism-based inhibition induced by a single dose of ecstasy (MDMA, 3,4-methylenedioxymethamphetamine) and evaluation of the tramadol ability as a probe drug. CYP2D1 is orthologous in rats to human CYP2D6 and was employed in the current study. Methods A total of 16 male rats were selected and divided into control and treatment groups. The control group did not receive MDMA, while rats in the treatment group received a single dose of MDMA (1 mg/kg) and were subsequently divided into groups that were tested at 1 h, 10 days or 30 days post-administration. The rats were subjected to liver perfusion with Krebs-Heinslet buffer containing tramadol for 60 min and the tramadol and M1 levels were determined by HPLC-fluorescence. Results The enzymatic activity of CYP2D1 for the 1-h group decreased significantly when compared with the control group (p<0.05). Moreover, enzymatic activity increased non-significantly in the 10- and 30-day groups in comparison with the control group. The concentration and AUC0−60 of tramadol increased in the 1-h and 10-day groups when compared with the control group but decreased in the 30-day group; however, none of these changes was statistically significant (p>0.05). The M1 metabolic ratio in the 1-h group decreased significantly when compared with the control group (p<0.05). The M1 metabolic ratio of the 10-day group increased and of the 30-day group decreased, but neither of these changes were significant. Conclusions Regardless of the genotype, the enzymatic activity of rat CYP2D1 recovered by 10 days post-administration of MDMA. It appears that tramadol, irrespective of its stereoselectivity, is not able to appraise rat hepatic CYP2D1 activity. It can be extrapolated that tramadol is a not suitable probe drug for human hepatic CYP2D1 because CYP2D1 in rats is orthologous to human CYP2D6. Further animal and human studies are required to confirm this hypothesis.

Diminished inhibitory impact of ZnO nanoparticles on anaerobic fermentation by the presence of TiO2 nanoparticles: Phenomenon and mechanism

Engineered nanoparticle materials (ENMs) are widely and increasingly produced and employed in many sectors. The use of diverse ENMs potentially leads to the release of multiple ENMs into the environment. These ENMs after discharge will be end in wastewater treatment plant and present in sludge. This work investigated the effect of multi-ENMs systems of ZnO and TiO2 on sludge anaerobic fermentation and the related toxicity mechanism. Results revealed that the toxicity of ZnO ENMs on anaerobic fermentation was reduced in the presence of TiO2 ENMs. Investigation on the change of free Zn2+ and reactive oxygen species (OH and H2O2) suggested that both of free Zn2+ and ROS contributed to the toxicity mechanism. Zn2+ decrease was the main reason for the reduced toxicity in multi-ENMs systems. ROS mainly led to the reduction of cell viability in anaerobic fermentation systems. The presence of TiO2 in the multi-ENMs systems promoted the recovery of enzyme activity, cell viability and bacteria abundance.

Effects of allantoin and dimethyl sulfoxide on the thermal aggregation of lysozyme

Allantoin is used to suppress protein aggregation without decreasing the melting temperature. However, the solubility of allantoin in water or buffer solutions is too low (approximately 30 mM at ambient temperature) to be used as a protein aggregation suppressor in various situations. Here we show that a high-concentration solution of allantoin in neat dimethyl sulfoxide (DMSO) is useful as a stock solution for the additive that controls protein aggregation. The allantoin concentration from 10 to 100 mM in 10% (v/v) DMSO significantly suppressed the thermal aggregation of hen egg white lysozyme as a model protein, with increasing allantoin concentration. The residual activity of lysozyme in 10% DMSO and 100 mM allantoin after heating at 90 °C for 10 min was increased >3-fold compared to that without allantoin. Thus, it was concluded that allantoin in DMSO is an effective stock solution for practical application in enhancing the recovery of enzymatic activity and suppressing the formation of small aggregate of protein.

Immobilization of cellulase on a core-shell structured metal-organic framework composites: Better inhibitors tolerance and easier recycling

For the first time, cellulase was successfully immobilized on a magnetic core-shell metal-organic framework (MOF) material, UIO-66-NH2. The as-prepared immobilized cellulase demonstrated a high protein loading efficiency of 126.2 g/g support and a high enzyme activity recovery of 78.4%. Cellulase immobilized on magnetic UIO-66-NH2 exhibited a superior performance in terms of pH stability, thermal stability and catalytic efficiency compared to its free form. Notably, immobilized cellulase could be recycled for up to 5 consecutive runs. Furthermore, compared to free cellulase, immobilized cellulase showed better tolerance to formic acid and vanillin, two typical inhibitors found in lignocellulosic prehydrolysates. In the presence of 5 g/L of formic acid and vanillin, immobilized cellulase demonstrated 16.8% and 21.5% higher activity than free enzyme, respectively, and its improvement in hydrolysis yield was 18.7% and 19.6% respectively. This is firstly confirmed that immobilization can alleviate the inhibitory effects of certain pretreatment inhibitors on cellulase.

Optimization of Down-Stream for Cellulases Produced Under Solid-State Fermentation of Coffee Husk

This work systematically studies the downstream process of the solid state fermentation (SSF) of a mixture of coffee husk and wood chips, inoculated with compost, for cellulase production. Downstream of SSF (at pilot scale) remains as one of the less studied stages of the process, being critical in technical, environmental and economic terms. In this study, the specific downstream points considered were: (i) enzyme extraction yield, in terms of extraction ratio solid:solvent, agitation mode and solvent type; (ii) enzymatic activity recovery of the lyophilised extract and (iii) efficiency of consecutive extractions. Results indicate a maximum activity recovery of 108 ± 30% in the extraction performed at ratio 1:5 solid-solvent, in static mode and with distilled water. Statistical analysis revealed a high dispersion of the results and needs to be considered to extract consistent conclusions in any downstream of SSF. Lyophilisation demonstrated to be an adequate technology for enzymatic activity preservation. Regarding consecutive extractions, yield recovery in the first and second extraction maintain a similar value. In a framework of a zero-waste enzyme production process, different strategies have been tested for the remaining solid after extraction. Respirometric tests reveal that it is possible to aerobically stabilize the remaining solid obtaining a compost like material, whereas anaerobic digestion resulted in low methane yields (51 ± 3 mL methane g−1 VS).

Covalent immobilization of cytochrome P450 BM3 (R966D/W1046S) on glutaraldehyde activated SPIONs

AbstractSelective hydroxylation of the C‐H bond of saturated hydrocarbon chains at room temperature is the signature of an invaluable biocatalyst, cytochrome P450 BM3 from Bacillus megaterium. Despite this remarkable ability, because of the enzyme's inherent low stability and dependence on electron supply by expensive NADPH, developing stable and economic BM3 systems is a challenging subject. To improve BM3 stability, facilitate its reuse, and reduce the process cost, this study suggests covalent immobilization of R966D/W1046S P450 BM3 on glutaraldehyde pre‐activated super paramagnetic iron oxide nanoparticles (SPIONs). This double mutant consumes less expensive cofactors like NADH and BNAH and its immobilization on magnetic support facilitates its separation and reuse. Free and immobilized enzyme performances were evaluated by 10‐pNCA hydroxylation and BM3 selectivity (hydroxylation at ω (1–3) positions of a fatty acid) was confirmed in a reaction involving myristic acid. The enzyme activity recovery was up to 60 % with 100 % enzyme binding efficiency. BM3‐SPIONs were easily separated from the reaction medium by applying a magnet, and recycled for 5 times, after which they could still present half of their initial activity. The enzyme storage stability was significantly improved: after one month of storage at 4 °C, the immobilized enzyme showed 80 % residual activity toward NADH while the soluble enzyme was inactive after a week. Binding an enzyme to fabricated SPIONs is a promising technique to increase enzyme stability and prevent downstream contamination in biocatalytic processes. In this context, BM3‐SPIONs can be a practical model system in cost‐effective large‐scale applications of such enzymes.

TRAF2 and TRAF5 associated with the signal transducing receptor gp130 limit IL-6-driven transphosphorylation of JAK1 through the inhibition of proximal JAK-JAK interaction.

Tumor necrosis factor receptor-associated factor 2 (TRAF2) and TRAF5 constitutively bind to glycoprotein 130 kDa (gp130) and inhibit IL-6-driven activation of signal transducer and activator of transcription 3 (STAT3) in CD4+ T cells, which limits the differentiation of pro-inflammatory IL-17-producing helper T cells that require IL-6-receptor (IL-6R) signals for their development. However, it is not known how the interaction between TRAF and gp130 negatively regulates STAT3 activity in the IL-6R complex. We hypothesized that TRAF proteins associated with gp130 might limit the activation of Janus kinase that is needed for the activation of STAT3. To test this, we transfected HEK293T cells to express gp130 and TRAF2 or TRAF5 together with two chimeric JAK1 proteins combined with either the N-terminal or the C-terminal protein fragment of firefly luciferase. Using this luciferase fragment complementation system, we found that the recovery of luciferase enzyme activity was coincident with proximal JAK1-JAK1 interaction and phosphorylation of JAK1 in the IL-6R complex and that the expression of TRAF protein significantly inhibited the recovery of luciferase activity. The binding of TRAF to gp130 via the C-terminal TRAF domain was essential for the inhibition. In accordance with this, upon stimulation of endogenous gp130 with a complex of IL-6 and IL-6R, Traf5-/- CD4+ T cells displayed significantly higher amounts of phosphorylated JAK1 than did their wild-type counterparts. Therefore, our results demonstrate that gp130-associated TRAF2 and TRAF5 inhibit the interaction between two JAK proteins in the IL-6R complex that is essential for initiating the JAK-STAT signaling pathway.

Preparation of a novel nanobiocatalyst by immobilizing penicillin acylase onto magnetic nanocrystalline cellulose and its use for efficient synthesis of cefaclor

Magnetic nanocrystalline cellulose (MNCC) was prepared and used as an enzyme support for the immobilization of penicillin acylase (PA). A novel coupling agent, tri(hydroxymethyl)phosphine(THP) instead of the conventional glutaraldehyde(GA), was used as a crosslinker in this study. The obtained results showed that the immobilized PA with THP (PA-THP-MNCC) had high enzyme loading (172.3 mg/g) and activity recovery (77.6%) in the optimal preparation conditions, which were remarkably superior to those of the counterpart using GA (PA-GA-MNCC, 148.4 mg/g and 48.7%, respectively). Compared with free PA and PA-GA-MNCC, PA-THP-MNCC displayed a higher optimum pH and temperature, and manifested relatively higher enzyme-substrate affinity and catalytic efficiency. In addition, PA-THP-MNCC exhibited significantly enhanced stability. Furthermore, PA-THP-MNCC was successfully employed for synthesis of cefaclor, an important second-generation cephalosporin antibiotic, affording a significantly higher yield of 84% than that reported previously.

Immobilization of glycerol dehydrogenase and NADH oxidase for enzymatic synthesis of 1,3‐dihydroxyacetone with in situ cofactor regeneration

AbstractBACKGROUNDNicotinamide cofactor‐dependent oxidoreductases have been widely applied to the bioproduction of varieties of useful compounds. Efficient cofactor regeneration is often required for these oxidoreductase‐catalyzed reactions. Herein, enzymatic production of 1, 3‐dihydroxyacetone (DHA) via immobilized enzymes with in situ cofactor regeneration was reported.RESULTSGlycerol dehydrogenase (GDH) and NADH oxidase (Nox) were immobilized on two carriers: macroporous resin and epoxy functionalized magnetic nanoparticles (EFMN). Higher enzyme loading capacity and activity recovery were achieved on EFMN. A high immobilization yield of 90% and >70% recovery activity were obtained for both enzymes using EFMN as carriers. Besides, both immobilized enzymes showed enhanced pH stability and temperature tolerance. Co‐immobilized and mixed immobilized enzymes were evaluated for synthesis of DHA, and mixed immobilized enzymes showed higher catalytic rate. The mixed reaction system also was investigated in view of various operational factors. Under the optimal conditions, a high DHA concentration with mixed immobilized enzymes system can reach up to 3.5 mmol L–1, which was about four times higher than that without cofactor regeneration system.CONCLUSIONThe immobilized GDH and Nox on magnetic nanoparticles showed enhanced stabilities. A cofactor regeneration system with immobilized enzymes could be utilized for efficient production of DHA from inexpensive substrates. © 2018 Society of Chemical Industry

Preparation, characterization and catalytic behavior of pectinase covalently immobilized onto sodium alginate/graphene oxide composite beads

Pectinase was immobilized onto sodium alginate/graphene oxide beads via amide bonds by using N,N′-dicyclohexylcarbodiimide/N-hydroxysuccinimide as the activating agent. The immobilized pectinase was characterized by Fourier transform infrared spectra and scanning electron microscopy analyses. Immobilization conditions were optimized by Box–Behnken design and the response surface method. The activity of the immobilized pectinase prepared under optimal conditions reached 1236.86 ± 40.21 U/g, with an enzyme activity recovery of 83.5%. The optimal pH of free pectinase was 4.5, while that of immobilized pectinase was shifted to 4.0. The optimal temperature of immobilized pectinase was increased to 60 °C, which was 10 °C higher than that of free form. Furthermore, the immobilized pectinase possessed a superior thermal stability and storage stability to those of free pectinase. Reusability studies indicated that the immobilized pectinase retained 73% of initial activity after six times cycles. Due to these good properties, such immobilized pectinase may find application in food industry.

Research of a new metal chelating carrier preparation and papain immobilization

A new type of magnetic metal chelating carrier (PCMM-IDA-Cu2+) was prepared for the immobilization of papain, using chitosan as raw material, nano Fe3O4 as magnetic material, SiO2 as porogen, iminodiacetic acid (IDA) as a chelating ligand, and binding with transition metal ion (Cu2+). The resulting products were well characterized by optical microscopy, scanning electron microscopy (SEM), vibrating sample magnetometer (VSM), Fourier transform infrared spectroscopy (FTIR) and thermogravimetric analysis (TGA). The BBD (Box-Behnken Design) of RSM (Response Surface Methodology) was applied to analyze the optimum enzyme immobilization conditions. The results showed that the enzyme immobilization capacity was 94.18mg/g of PCMM-IDA-Cu2+, with 7.976U/mg of relative immobilized enzyme activity under the optimum conditions (pH6.73, 1.56mg enzyme/15.0mg carrier, 30.9°C), and the recovery of enzyme activity was reached 87.21%. Compared with the free papain, the immobilized papain displayed enhanced enzyme activity, superior enzymatic properties, good operational stability and reusability. It is worth mentioning that the novel carriers exhibited selectively biological adsorption capacity, and this technique is an alternative method for the immobilization of enzyme, making the process more efficient and economic.

Temperature-induced recovery of a bioactive enzyme using polyglycerol dendrimers: correlation between bound water and protein interaction

Enzyme application has gained importance over the past decade in bioprocess, biomedical, and pharmaceutical fields. We found that polyglycerol dendrimers (PGDs), which are biocompatible molecules, can recover alcohol dehydrogenase (ADH) from aqueous solution under elevated temperature. A low concentration of PGD (5 wt.%) is sufficient for the recovery of high enzymatic activity, although a high concentration (25–75 wt.%) of glycerol is generally required to stabilize ADH. The enzymatic activity of ADH in suspension with PGDs is over 60% but it is only 10% in that with glycerol. The results of osmolarity and spin-lattice relaxation time (T1) of water measurements in the presence of PGDs suggest that increased amounts of bound water to PGD molecules trigger aggregation along with the direct interaction with ADH. PGDs therefore represent good potential additives for direct recovery of enzymes from aqueous solutions.

Magnetic ZIF-8/cellulose/Fe3O4 nanocomposite: preparation, characterization, and enzyme immobilization

BackgroundThe ZIF-8-coated magnetic regenerated cellulose-coated nanoparticles (ZIF-8@cellu@Fe3O4) were successfully prepared and characterized. The result showed that ZIF-8 was successfully composited on to the surface of the cellulose-coated Fe3O4 nanoparticles by co-precipitation method. Moreover, the glucose oxidase (GOx, from Aspergillus niger) was efficiently immobilized by the ZIF-8@Cellu@Fe3O4 nanocarriers with enhanced catalytic activities. The enzyme loading was 94.26 mg/g and the enzyme activity recovery was more than 124.2%. This efficiently immobilized enzyme exhibits promising applications in biotechnology, diagnosis, biosensing, and biomedical devices.ConclusionsA new core–shell magnetic ZIF-8/cellulose nanocomposite (ZIF-8@Cellu@Fe3O4) was fabricated and structurally characterized. Glucose oxidase (GOx) was successfully immobilized by the biocompatible ZIF-8@Cellu@Fe3O4 with high protein loading (94.26 mg/g) and enhanced relative activity recovery (124.2%).

Identification and analysis of the reactive metabolites related to the hepatotoxicity of safrole

1. Safrole is the main component of the volatile oil in Xixin, which has a strong antifungal effect. However, safrole has been shown to be associated with the development of hepatocellular carcinoma. Methylenedioxyphenyl and allyl-benzene substructures of safrole may cause a mechanism-based inhibition (MBI) of CYP450 enzymes (CYPs) and produce reactive metabolites (RMs), resulting in inhibition of enzyme activity and toxic effects.2. Based on the experiments of CYPs cocktail screening, glutathione (GSH) capture and the IC50 data, we found that safrole had an inhibitory effect on CYP1A2. The test of enzyme activity recovery when adding GSH may help to verify the MBI of safrole.3. Two metabolites, 1,2-dihydroxy-4-allylbenzene (M1) and 1′-hydroxy safrole (M2) could be captured by GSH. The ultra performance liquid chromatography - tandem mass spectrometer (UPLC-MS/MS) method was used to identify the RMs through a detailed characterization of the safrole cleavage processes and the GSH-M1 adduct. The RMs identified are quinone and its tautomer. Thus, preliminary conclusion can be obtained that safrole is a mechanism-based inhibitor of CYP1A2.4. The cleavage process of the GSH-M1/M2 adduct was analyzed in further detail. We believe the safrole hepatotoxicity mechanism is related to the RMs mediated by CYP1A2. This work provides important information on predicting in vivo drug induced liver injury.

A simple method for purification of bromelain in a thermosensitive triblock copolymer-based protection system and recycling of phase components

ABSTRACTIn the work, we chose stem bromelain as a model to investigate the storage and purification of bromelain from pineapple peel. Extraction of bromelain from pineapple peel using a two-stage aqueous two-phase extraction system composed of a thermoseparating copolymer EOPOEO and K2HPO4. Bromelain predominantly partitioned to the EOPOEO-rich phase and then re-extract to the top dilute phase. The recovery of enzyme activity (68.6%) and purification factor (6.53) were determined under optimum conditions. The EOPOEO-rich phase and salt were recycled, and the recovery of enzyme activity could reach up to 60%. This method has been proved to obtain highly purified and stable bromelain.

Effect of Controlled Ice Nucleation on Stability of Lactate Dehydrogenase During Freeze-Drying

Several controlled ice nucleation techniques have been developed to increase the efficiency of the freeze-drying process as well as to improve the quality of pharmaceutical products. Owing to the reduction in ice surface area, these techniques have the potential to reduce the degradation of proteins labile during freezing. The objective of this study was to evaluate the effect of ice nucleation temperature on the in-process stability of lactate dehydrogenase (LDH). LDH in potassium phosphate buffer was nucleated at −4°C, −8°C, and −12°C using ControLyo™ or allowed to nucleate spontaneously. Both the enzymatic activity and tetramer recovery after freeze-thawing linearly correlated with product ice nucleation temperature (n = 24). Controlled nucleation also significantly improved batch homogeneity as reflected by reduced inter-vial variation in activity and tetramer recovery. With the correlation established in the laboratory, the degradation of protein in manufacturing arising from ice nucleation temperature differences can be quantitatively predicted. The results show that controlled nucleation reduced the degradation of LDH during the freezing process, but this does not necessarily translate to vastly superior stability during the entire freeze-drying process. The capability of improving batch homogeneity provides potential advantages in scaling-up from lab to manufacturing scale.