Protein-protein interaction and site prediction using transfer learning.

The advanced language models have enabled us to recognize protein-protein interactions (PPIs) and interaction sites using protein sequences or structures. Here, we trained the MindSpore ProteinBERT (MP-BERT) model, a Bidirectional Encoder Representation from Transformers, using protein pairs as inputs, making it suitable for identifying PPIs and their respective interaction sites. The pretrained model (MP-BERT) was fine-tuned as MPB-PPI (MP-BERT on PPI) and demonstrated its superiority over the state-of-the-art models on diverse benchmark datasets for predicting PPIs. Moreover, the model's capability to recognize PPIs among various organisms was evaluated on multiple organisms. An amalgamated organism model was designed, exhibiting a high level of generalization across the majority of organisms and attaining an accuracy of 92.65%. The model was also customized to predict interaction site propensity by fine-tuning it with PPI site data as MPB-PPISP. Our method facilitates the prediction of both PPIs and their interaction sites, thereby illustrating the potency of transfer learning in dealing with the protein pair task.

Cadmium-absorptive Bacillus vietnamensis 151-6 reduces the grain cadmium accumulation in rice (Oryza sativa L.): Potential for cadmium bioremediation.

Microbial bioremediation of heavy metal-polluted soil is a promising technique for reducing heavy metal accumulation in crops. In a previous study, we isolated Bacillus vietnamensis strain 151-6 with a high cadmium (Cd) accumulation ability and low Cd resistance. However, the key gene responsible for the Cd absorption and bioremediation potential of this strain remains unclear. In this study, genes related to Cd absorption in B. vietnamensis 151-6 were overexpressed. A thiol-disulfide oxidoreductase gene (orf4108) and a cytochrome C biogenesis protein gene (orf4109) were found to play major roles in Cd absorption. In addition, the plant growth-promoting (PGP) traits of the strain were detected, which enabled phosphorus and potassium solubilization and indole-3-acetic acid (IAA) production. Bacillus vietnamensis 151-6 was used for the bioremediation of Cd-polluted paddy soil, and its effects on growth and Cd accumulation in rice were explored. The strain increased the panicle number (114.82%) and decreased the Cd content in rice rachises (23.87%) and grains (52.05%) under Cd stress, compared with non-inoculated rice in pot experiments. For field trials, compared with the non-inoculated control, the Cd content of grains inoculated with B. vietnamensis 151-6 was effectively decreased in two cultivars (low Cd-accumulating cultivar: 24.77%; high Cd-accumulating cultivar: 48.85%) of late rice. Bacillus vietnamensis 151-6 encoded key genes that confer the ability to bind Cd and reduce Cd stress in rice. Thus, B. vietnamensis 151-6 exhibits great application potential for Cd bioremediation.

Combined assembly of long and short sequencing reads improve the efficiency of exploring the soil metagenome.

BACKGROUND: Advances in DNA sequencing technologies have transformed our capacity to perform life science research, decipher the dynamics of complex soil microbial communities and exploit them for plant disease management. However, soil is a complex conglomerate, which makes functional metagenomics studies very challenging. RESULTS: Metagenomes were assembled by long-read (PacBio, PB), short-read (Illumina, IL), and mixture of PB and IL (PI) sequencing of soil DNA samples were compared. Ortholog analyses and functional annotation revealed that the PI approach significantly increased the contig length of the metagenomic sequences compared to IL and enlarged the gene pool compared to PB. The PI approach also offered comparable or higher species abundance than either PB or IL alone, and showed significant advantages for studying natural product biosynthetic genes in the soil microbiomes. CONCLUSION: Our results provide an effective strategy for combining long and short-read DNA sequencing data to explore and distill the maximum information out of soil metagenomics.

The endophytic bacterium Bacillus koreensis 181-22 promotes rice growth and alleviates cadmium stress under cadmium exposure.

Recently, cadmium (Cd) contamination in paddy soils has become a highly concerning pollution problem. Endophytic microbes in rice not only affect the plant growth but also contribute to ion absorption by the roots. Therefore, they are a promising, ecologically sound means of reducing the Cd transport from soils to shoots and grains of the plant. In this study, a Cd-resistant endophytic bacterium, named 181-22, with high Cd absorption capacity (90.8%) was isolated from the roots of rice planting in heavily Cd-contaminated paddy soils and was identified as Bacillus koreensis CGMCC 19,468. The strain significantly increased fresh weight of roots and shoots (44.4% and 42.7%) and dry weight of roots and shoots (71.3% and 39.9%) and decreased Cd content in the rice roots (12.8%), shoots (34.3%), and grains (39.1%) under Cd stress compared to uninoculated plant by colonizing rice roots via seed inoculation. Moreover, colonization of 181-22 reprogrammed rice physiology to alleviate Cd stress by increasing pigment and total protein content, regulating Cd-induced oxidative stress enzymes such as superoxide dismutase and catalase and reducing malondialdehyde. Thus, B. koreensis 181-22 has the potential to protect rice against Cd stress and can be used as a biofertilizer to bioremediate paddy soils contaminated with Cd. KEY POINTS: • Bacillus koreensis 181-22 colonized the inside of rice roots at high numbers via seed inoculation. • B. koreensis 181-22 promoted rice growth and decreased Cd accumulation in grains. • B. koreensis 181-22 regulated the physiological response to alleviated Cd stress in rice.

An operon consisting of a P-type ATPase gene and a transcriptional regulator gene responsible for cadmium resistances in Bacillus vietamensis 151-6 and Bacillus marisflavi 151-25.

BACKGROUND: Cadmium (Cd) is a severely toxic heavy metal to most microorganisms. Many bacteria have developed Cd2+ resistance. RESULTS: In this study, we isolated two different Cd2+ resistance Bacillus sp. strains, Bacillus vietamensis 151-6 and Bacillus marisflavi 151-25, which could be grown in the presence of Cd2+ at concentration up to 0.3 mM and 0.8 mM, respectively. According to the genomic sequencing, transcriptome analysis under cadmium stress, and other related experiments, a gene cluster in plasmid p25 was found to be a major contributor to Cd2+ resistance in B. marisflavi 151-25. The cluster in p25 contained orf4802 and orf4803 which encodes an ATPase transporter and a transcriptional regulator protein, respectively. Although 151-6 has much lower Cd2+ resistance than 151-25, they contained similar gene cluster, but in different locations. A gene cluster on the chromosome containing orf4111, orf4112 and orf4113, which encodes an ATPase transporter, a cadmium efflux system accessory protein and a cadmium resistance protein, respectively, was found to play a major role on the Cd2+ resistance for B. vietamensis 151-6. CONCLUSIONS: This work described cadmium resistance mechanisms in newly isolated Bacillus vietamensis 151-6 and Bacillus marisflavi 151-25. Based on homologies to the cad system (CadA-CadC) in Staphylococcus aureus and analysis of transcriptome under Cd2+ induction, we inferred that the mechanisms of cadmium resistance in B. marisflavi 151-25 was as same as the cad system in S. aureus. Although Bacillus vietamensis 151-6 also had the similar gene cluster to B. marisflavi 151-25 and S. aureus, its transcriptional regulatory mechanism of cadmium resistance was not same. This study explored the cadmium resistance mechanism for B. vietamensis 151-6 and B. marisflavi 151-25 and has expanded our understanding of the biological effects of cadmium.

Identification of a chitosanase from the marine metagenome and its molecular improvement based on evolution data.

Chitooligosaccharides have important application value in the fields of food and agriculture. Chitosanase can degrade chitosan to obtain chitooligosaccharides. The marine metagenome contains many genes related to the degradation of chitosan. However, it is difficult to mine valuable genes from large gene resources. This study proposes a method to screen chitosanases directly from the marine metagenome. Chitosanase gene chis1754 was identified from the metagenome and heterologously expressed in Escherichia coli. The optimal temperature and pH of CHIS1754 were 55 °C and 5.5, respectively. A mutant, CHIS1754T, with 15 single point mutations designed based on molecular evolution data was also expressed in E. coli. The results indicated that the thermal stability of CHIS1754T was significantly improved, as the Tm showed an increase of ~ 7.63 °C. Additionally, the kcat/Km of CHIS1754T was 4.8-fold higher than that of the wild type. This research provides new theories and foundations for the excavation, modification, and industrial application of chitosanases. KEY POINTS: A chitosanase gene, chis1754, was firstly identified from marine metagenome. A multi-site mutant was designed to improve enzyme stability and activity. The kcat/Kmof the designed mutant was 4.8-fold higher than that of the wild type.

Identification and characterization of sequence signatures in the Bacillus subtilis promoter Pylb for tuning promoter strength.

OBJECTIVE: To thoroughly characterize the Pylb promoter and identify the elements that affect the promoter activity. RESULT: The sequences flanking the - 35 and - 10 box of the Pylb promoter were divided into six segments, and six random-scanning mutant promoter libraries fused to an enhanced green fluorescent protein EGFP were made and analyzed by flow cytometry. Our results showed that the four nucleotides flanking the - 35 box could mostly influence the promoter activity, and this influence was related to the GC content. The promoters mutated in these regions were successfully used for expressing the gene ophc2 encoding organophosphorus hydrolase (OPHC2) and the gene katA encoding catalase (KatA). CONCLUSION: Our work identified and characterized the sequence signatures of the Pylb promoter that could tune the promoter strength, providing further information for the potential application of this promoter. Meanwhile, the sequence signatures have the potential to be used for tuning gene expression in enzyme production, metabolic engineering, and synthetic biology.

Effect of rare codons in C-terminal of green fluorescent protein on protein production in Escherichia coli.

In the previous study, the results on two interesting egfp genes indicated that the expressed eGFP production of egfp-codon containing multiple rare codons was 2.3-fold than that of egfp-genscript with mainly high-frequency-usage codons. Therefore, the rare codons also play important roles for the functional expression of genes and it is interesting to know which rare codons in the egfp affect the functional expression of eGFP. In this study, the structure-guided SCHEMA recombination method and site-specific mutagenesis were proposed to detect the contribution of the rare codons on the functional expression of eGFP. The 12 chimeric egfps were generated from egfp-codon and egfp-genscript by the software SCHEMA. The results indicated that it was the rare codons in the C-terminal coding region (residues from 147 to 239) of eGFP resulting in the higher expression levels in Escherichia coli. The single and multiple point mutations also indicated that the presence of rare codons in 3' coding regions of egfp could enhance the functional expression of eGFP in E. coli. Therefore, the gene sequence on the C-terminal could also affect its functional expression and the strategy of substituting rare codons into coding sequences might be an effective method for increasing heterologous proteins in the host.

Presyncodon, a Web Server for Gene Design with the Evolutionary Information of the Expression Hosts.

In the natural host, most of the synonymous codons of a gene have been evolutionarily selected and related to protein expression and function. However, for the design of a new gene, most of the existing codon optimization tools select the high-frequency-usage codons and neglect the contribution of the low-frequency-usage codons (rare codons) to the expression of the target gene in the host. In this study, we developed the method Presyncodon, available in a web version, to predict the gene code from a protein sequence, using built-in evolutionary information on a specific expression host. The synonymous codon-usage pattern of a peptide was studied from three genomic datasets (Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae). Machine-learning models were constructed to predict a selection of synonymous codons (low- or high-frequency-usage codon) in a gene. This method could be easily and efficiently used to design new genes from protein sequences for optimal expression in three expression hosts (E. coli, B. subtilis, and S. cerevisiae). Presyncodon is free to academic and noncommercial users; accessible at http://www.mobioinfor.cn/presyncodon_www/index.html.

Enhancing the soluble expression of an amylase in Escherichia coli by the mutations related to its domain interactions.

The sequence and structure of the target protein exert a marked effect on its soluble expression in Escherichia coli. The effects of the mutation of an amylase isolated from Bacillus licheniformis (BLA) on its soluble expression in E. coli were investigated. A random mutation library of BLA was constructed to screen for mutations that resulted in enhanced soluble expression in E. coli. Two interesting mutations (A390I and D401V) were identified, which are located at the interaction surface between the A and C domains of BLA. The A390I mutation enhanced soluble BLA expression by 2.0-fold compared to wild type, while D401V decreased soluble expression 160-fold. Structural analysis revealed that A390 and D401 residues could affect the interaction between the A and C domains of BLA. Therefore, soluble expression of the target protein in E. coli could be affected by introduction of a mutation in the protein sequence.

Enhanced secretion of a methyl parathion hydrolase in Pichia pastoris using a combinational strategy.

BACKGROUND: Although Pichia pastoris has been successfully used to produce various recombinant heterologous proteins, the efficiency varies. In this study, we used methyl parathion hydrolase (MPH) from Ochrobactrum sp. M231 as an example to study the effect of protein amino acid sequence on secretion from P. pastoris. RESULTS: The results indicated that the protein N-terminal sequence, the endoplasmic reticulum (ER) retention signal (KKXX) at the protein C-terminus, and the acidic stability of the protein could affect its secretion from P. pastoris. Mutations designed based on these sequence features markedly improved secretion from P. pastoris. In addition, we found that the secretion properties of a protein can be cumulative when all of the above strategies are combined. The final mutant (CHBD-DQR) designed by combining all of the strategies greatly improved secretion and the secreted MPH activity of CHBD-DQR was enhanced up to 195-fold compared with wild-type MPH without loss of catalytic efficiency. CONCLUSIONS: These results demonstrate that the secretion of heterologous proteins from P. pastoris could be improved by combining changes in multiple protein sequence features.

Harnessing microbial interactions with rice: Strategies for abiotic stress alleviation in the face of environmental challenges and climate change.

Rice, which feeds more than half of the world's population, confronts significant challenges due to environmental and climatic changes. Abiotic stressors such as extreme temperatures, drought, heavy metals, organic pollutants, and salinity disrupt its cellular balance, impair photosynthetic efficiency, and degrade grain quality. Beneficial microorganisms from rice and soil microbiomes have emerged as crucial in enhancing rice's tolerance to these stresses. This review delves into the multifaceted impacts of these abiotic stressors on rice growth, exploring the origins of the interacting microorganisms and the intricate dynamics between rice-associated and soil microbiomes. We highlight their synergistic roles in mitigating rice's abiotic stresses and outline rice's strategies for recruiting these microorganisms under various environmental conditions, including the development of techniques to maximize their benefits. Through an in-depth analysis, we shed light on the multifarious mechanisms through which microorganisms fortify rice resilience, such as modulation of antioxidant enzymes, enhanced nutrient uptake, plant hormone adjustments, exopolysaccharide secretion, and strategic gene expression regulation, emphasizing the objective of leveraging microorganisms to boost rice's stress tolerance. The review also recognizes the growing prominence of microbial inoculants in modern rice cultivation for their eco-friendliness and sustainability. We discuss ongoing efforts to optimize these inoculants, providing insights into the rigorous processes involved in their formulation and strategic deployment. In conclusion, this review emphasizes the importance of microbial interventions in bolstering rice agriculture and ensuring its resilience in the face of rising environmental challenges.

Engineering dual-functional and thermophilic BMHETase for efficient degradation of polyethylene terephthalate.

Polyethylene terephthalate (PET) biodegradation is hindered by the intermediates bis (2-hydroxyethyl) terephthalate (BHET) and mono (2-hydroxyethyl) terephthalate (MHET). BMHETase, a thermophilic hydrolase identified from the UniParc database, exhibits degradation activity towards both BHET and MHET. BMHETase showed higher activity on BHET than LCCICCG and FASTPETase at temperatures ranging from 50 to 70℃. To enhance its activity in degrading MHET, BMHETase was engineered to mimic Ideonella sakaiensis MHETase. The resulting 6-point mutant's activities on MHET and BHET were 8 and 2 times those of the WT, with both optimal temperatures increased by 5℃. This enhancement may be attributed to the BMHETase6M's intensified binding ability with MHET and enlarged binding pocket. When combined with LCCICCG, BMHETase6M achieved complete degradation of MHET in PET films to terephthalic acid, indicating broad application potential. These findings suggest that BMHETase6M holds promise as a candidate for enhancing PET biodegradation efficiency and plastic waste management.

The seed endophytic microbe Microbacterium testaceum M15 enhances the cold tolerance and growth of rice (Oryza sativa L.).

The potential of seed endophytic microbes to enhance plant growth and resilience is well recognized, yet their role in alleviating cold stress in rice remains underexplored due to the complexity of these microbial communities. In this study, we investigated the diversity of seed endophytic microbes in two rice varieties, the cold-sensitive CB9 and the cold-tolerant JG117. Our results revealed significant differences in the abundance of Microbacteriaceae, with JG117 exhibiting a higher abundance under both cold stress and room temperature conditions compared to CB9. Further analysis led to the identification of a specific cold-tolerant microbe, Microbacterium testaceum M15, in JG117 seeds. M15-inoculated CB9 plants showed enhanced growth and cold tolerance, with a germination rate increase from 40 % to 56.67 % at 14℃ and a survival rate under cold stress (4℃) doubling from 22.67 % to 66.67 %. Additionally, M15 significantly boosted chlorophyll content by over 30 %, increased total protein by 16.31 %, reduced malondialdehyde (MDA) levels by 37.76 %, and increased catalase activity by 26.15 %. Overall, our study highlights the potential of beneficial endophytic microbes like M. testaceum M15 in improving cold tolerance in rice, which could have implications for sustainable agricultural practices and increased crop productivity in cold-prone regions.

Enhancing the endo-activity of the thermophilic chitinase to yield chitooligosaccharides with high degrees of polymerization.

Thermophilic endo-chitinases are essential for production of highly polymerized chitooligosaccharides, which are advantageous for plant immunity, animal nutrition and health. However, thermophilic endo-chitinases are scarce and the transformation from exo- to endo-activity of chitinases is still a challenging problem. In this study, to enhance the endo-activity of the thermophilic chitinase Chi304, we proposed two approaches for rational design based on comprehensive structural and evolutionary analyses. Four effective single-point mutants were identified among 28 designed mutations. The ratio of (GlcNAc)3 to (GlcNAc)2 quantity (DP3/2) in the hydrolysates of the four single-point mutants undertaking colloidal chitin degradation were 1.89, 1.65, 1.24, and 1.38 times that of Chi304, respectively. When combining to double-point mutants, the DP3/2 proportions produced by F79A/W140R, F79A/M264L, F79A/W272R, and M264L/W272R were 2.06, 1.67, 1.82, and 1.86 times that of Chi304 and all four double-point mutants exhibited enhanced endo-activity. When applied to produce chitooligosaccharides (DP ≥ 3), F79A/W140R accumulated the most (GlcNAc)4, while M264L/W272R was the best to produce (GlcNAc)3, which was 2.28 times that of Chi304. The two mutants had exposed shallower substrate-binding pockets and stronger binding abilities to shape the substrate. Overall, this research offers a practical approach to altering the cutting pattern of a chitinase to generate functional chitooligosaccharides.

Improving the thermostability of methyl parathion hydrolase from Ochrobactrum sp. M231 using a computationally aided method.

Good protein thermostability is very important for the protein application. In this report, we propose a strategy which contained a prediction method to select residues related to protein thermal stability, but not related to protein function, and an experiment method to screen the mutants with enhanced thermostability. The prediction strategy was based on the calculated site evolutionary entropy and unfolding free energy difference between the mutant and wild-type (WT) methyl parathion hydrolase enzyme from Ochrobactrum sp. M231 [Ochr-methyl parathion hydrolase (MPH)]. As a result, seven amino acid sites within Ochr-MPH were selected and used to construct seven saturation mutagenesis libraries. The results of screening these libraries indicated that six sites could result in mutated enzymes exhibiting better thermal stability than the WT enzyme. A stepwise evolutionary approach was designed to combine these selected mutants and a mutant with four point mutations (S274Q/T183E/K197L/S192M) was selected. The Tm and T50 of the mutant enzyme were 11.7 and 10.2 °C higher, respectively, than that of the WT enzyme. The success of this design methodology for Ochr-MPH suggests that it was an efficient strategy for enhancing protein thermostability and suitable for protein engineering.

MECE: a method for enhancing the catalytic efficiency of glycoside hydrolase based on deep neural networks and molecular evolution.

The demand for high efficiency glycoside hydrolases (GHs) is on the rise due to their various industrial applications. However, improving the catalytic efficiency of an enzyme remains a challenge. This investigation showcases the capability of a deep neural network and method for enhancing the catalytic efficiency (MECE) platform to predict mutations that improve catalytic activity in GHs. The MECE platform includes DeepGH, a deep learning model that is able to identify GH families and functional residues. This model was developed utilizing 119 GH family protein sequences obtained from the Carbohydrate-Active enZYmes (CAZy) database. After undergoing ten-fold cross-validation, the DeepGH models exhibited a predictive accuracy of 96.73%. The utilization of gradient-weighted class activation mapping (Grad-CAM) was used to aid us in comprehending the classification features, which in turn facilitated the creation of enzyme mutants. As a result, the MECE platform was validated with the development of CHIS1754-MUT7, a mutant that boasts seven amino acid substitutions. The kcat/Km of CHIS1754-MUT7 was found to be 23.53 times greater than that of the wild type CHIS1754. Due to its high computational efficiency and low experimental cost, this method offers significant advantages and presents a novel approach for the intelligent design of enzyme catalytic efficiency. As a result, it holds great promise for a wide range of applications.

Rational redesign of thermophilic PET hydrolase LCCICCG to enhance hydrolysis of high crystallinity polyethylene terephthalates.

Polyethylene terephthalate (PET)-degrading enzymes represent a promising solution to the plastic pollution. However, PET-degrading enzymes, even thermophilic PETase, can effectively degrade low-crystallinity (∼8%) PETs, but exhibit weak depolymerization of more common, high-crystallinity (30-50%) PETs. Here, based on the thermophilic PETase, LCCICCG, we proposed two strategies for rational redesign of LCCICCG using the machine learning tool, Preoptem, combined with evolutionary analysis. Six single-point mutants (S32L, D18T, S98R, T157P, E173Q, N213P) were obtained that exhibit higher catalytic efficiency towards PET powder than wild-type LCCICCG at 75 °C. Additionally, the optimal temperature for degrading 39.07% crystalline PET increased from 65 °C in the wild-type LCCICCG to between 75 and 80 °C in the LCCICCG_I6M mutant that carries all six single-point mutations. Especially, the LCCICCG_I6M mutant has a significantly higher degradation effect on some commonly used bottle-grade plastic powders at 75-80 °C than that of wild type. The enzymatic digestion of ground 31.30% crystalline PET water bottles by LCCICCG_I6M yielded 31.91 ± 0.99 mM soluble products in 24 h, which was 3.64 times that of LCCICCG (8.77 ± 1.52 mM). Overall, this study provides a feasible route for engineering thermostable enzymes that can degrade high-crystallinity PET plastic.

Identification of the para-nitrophenol catabolic pathway, and characterization of three enzymes involved in the hydroquinone pathway, in Peudomonas sp. 1-7.

BACKGROUND: para-Nitrophenol (PNP), a priority environmental pollutant, is hazardous to humans and animals. However, the information relating to the PNP degradation pathways and their enzymes remain limited. RESULTS: Pseudomonas sp.1-7 was isolated from methyl parathion (MP)-polluted activated sludge and was shown to degrade PNP. Two different intermediates, hydroquinone (HQ) and 4-nitrocatechol (4-NC) were detected in the catabolism of PNP. This indicated that Pseudomonas sp.1-7 degraded PNP by two different pathways, namely the HQ pathway, and the hydroxyquinol (BT) pathway (also referred to as the 4-NC pathway). A gene cluster (pdcEDGFCBA) was identified in a 10.6 kb DNA fragment of a fosmid library, which cluster encoded the following enzymes involved in PNP degradation: PNP 4-monooxygenase (PdcA), p-benzoquinone (BQ) reductase (PdcB), hydroxyquinol (BT) 1,2-dioxygenase (PdcC), maleylacetate (MA) reductase (PdcF), 4-hydroxymuconic semialdehyde (4-HS) dehydrogenase (PdcG), and hydroquinone (HQ) 1,2-dioxygenase (PdcDE). Four genes (pdcDEFG) were expressed in E. coli and the purified pdcDE, pdcG and pdcF gene products were shown to convert HQ to 4-HS, 4-HS to MA and MA to ß-ketoadipate respectively by in vitro activity assays. CONCLUSIONS: The cloning, sequencing, and characterization of these genes along with the functional PNP degradation studies identified 4-NC, HQ, 4-HS, and MA as intermediates in the degradation pathway of PNP by Pseudomonas sp.1-7. This is the first conclusive report for both 4-NC and HQ- mediated degradation of PNP by one microorganism.

Improving the acidic stability of a methyl parathion hydrolase by changing basic residues to acidic residues.

The acidic stability of a methyl parathion hydrolase (Ochr-MPH) was improved by selectively changing basic amino acids to acidic ones. Mutation sites were selected based on the position-specific amino acid replacement probabilities (more than or equal to 0.2) and the entropy of each site (more than or equal to 0.8). Three mutants (K208E, K277D, and K208E/K277D) were more stable than the wild-type (WT). Their half-lives at pH 5.0 were 64, 68, 65 min, respectively, whereas that of WT was 39 min. The acidic stability of proteins may therefore be improved by changing selected basic amino acid residues to acidic ones.