Zhihao Sun

Fermentative production of succinic acid from straw hydrolysate by Actinobacillus succinogenes

In this work, straw hydrolysates were used to produce succinic acid by Actinobacillus succinogenes CGMCC1593 for the first time. Results indicated that both glucose and xylose in the straw hydrolysates were utilized in succinic acid production, and the hydrolysates of corn straw was better than that of rice or wheat straw in anaerobic fermentation of succinic acid. However, cell growth and succinic acid production were inhibited when the initial concentration of sugar, which was from corn straw hydrolysate (CSH), was higher than 60 g l(-1). In batch fermentation, 45.5 g l(-1) succinic acid concentration and 80.7% yield were attained after 48 h incubation with 58 g l(-1) of initial sugar from corn straw hydrolysate in a 5-l stirred bioreactor. While in fed-batch fermentation, concentration of succinic acid achieved 53.2 g l(-1) at a rate of 1.21 g l(-1) h(-1) after 44 h of fermentation. Our work suggested that corn straw could be utilized for the economical production of succinic acid by A. succinogenes.

Arginine deiminase, a potential anti-tumor drug.

Arginine deiminase (ADI; EC 3.5.3.6), an arginine-degrading enzyme, has been studied as a potential anti-tumor drug for the treatment of arginine-auxotrophic tumors, such as hepatocellular carcinomas (HCCs) and melanomas. Studies with human lymphatic leukemia cell lines further suggest that ADI is a potential anti-angiogenic agent and is effective in the treatment of leukemia. For instance ADI-PEG-20, patented by Pheonix Pharmacologic Inc., is currently in clinical trials for the treatment of HCC (Phase II/III) and melanoma (Phase I/II). This review summarizes results on recombinant expression, structural analysis, PEG (polyethylene glycerol) modification, in vivo anti-cancer activities, and clinical studies of ADI. Discussions on heterogeneous expression of ADI, directed evolution for improving enzymatic properties, and HSA-fusion for increased in vivo activity conclude this review.

Succinic acid production from corn stover by simultaneous saccharification and fermentation using Actinobacillus succinogenes

Simultaneous saccharification and fermentation (SSF) technique was applied for succinic acid production by Actinobacillus succinogenes in a 5-l stirred bioreactor with corn stover as the raw material. The process parameters of SSF, including corn stover pretreatment condition, substrate concentration, enzyme loading and fermentation temperature were investigated. Results indicated that pretreating corn stover with diluted alkaline was beneficial for the succinic acid production, and succinic acid yield could be significantly increased when adding the cellulase supplemented with cellobiase. The maximal succinic acid concentration and yield could reach 47.4 g/l and 0.72 g/g-substrate, respectively. The corresponding operation conditions were summarized as follows: SSF operation at 38 °C for 48 h, diluted alkaline pretreated corn stover as substrate with concentration of 70 g/l, enzyme loading of 20FPU cellulase and 10 U cellobiase per gram substrate. This result suggested an industrial potential of succinic acid production by using SSF and corn stover.

Continuous butanol fermentation from inexpensive sugar-based feedstocks by Clostridium saccharobutylicum DSM 13864

Corn stover hydrolysate (CSH) and cane molasses were studied for butanol fermentation by Clostridium saccharobutylicum DSM 13864 in continuous fermentation. Using cane molasses as substrate, solvent of 13.75 g/L (butanol 8.65 g/L) and productivity of 0.439 g/L/h were achieved in a four-stage continuous fermentation at a gradient dilution mode of 0.15-0.15-0.125-0.1 h(-1). In continuous fermentation using CSH as substrate, total solvent titer of 11.43 g/L (butanol 7.81 g/L) and productivity of 0.429 g/L/h were reached at a dilution rate of 0.15 h(-1), and the steady process was continuously operated for 220 h without compromise in solvent titer.

Scalable biocatalytic synthesis of optically pure ethyl (R)-2-hydroxy-4-phenylbutyrate using a recombinant E. coli with high catalyst yield

Ethyl (R)-2-hydroxy-4-phenylbutanoate [(R)-HPBE] is a versatile and important chiral intermediate for the synthesis of angiotensin-converting enzyme (ACE) inhibitors. Recombinant E. coli strain coexpressing a novel NADPH-dependent carbonyl reductase gene iolS and glucose dehydrogenase gene gdh from Bacillus subtilis showed excellent catalytic activity in (R)-HPBE production by asymmetric reduction. IolS exhibited high stereoselectivity (>98.5% ee) toward α-ketoesters substrates, whereas fluctuant ee values (53.2-99.5%) for β-ketoesters with different halogen substitution groups. Strategies including aqueous/organic biphasic system and substrate fed-batch were adopted to improve the biocatalytic process. In a 1-L aqueous/octanol biphasic reaction system, (R)-HPBE was produced in 99.5% ee with an exceptional catalyst yield (g product/g catalyst) of 31.7 via bioreduction of ethyl 2-oxo-4-phenylbutyrate (OPBE) at 330 g/L.

Directed Evolution of an Antitumor Drug (Arginine Deiminase PpADI) for Increased Activity at Physiological pH

Arginine deiminase (ADI; EC 3.5.3.6) has been studied as a potential antitumor drug for the treatment of arginine‐auxotrophic tumors, such as hepatocellular carcinomas (HCCs) and melanomas. Studies with human lymphatic leukemia cell lines confirmed that ADI is an antiangiogenic agent for treating leukemia. The main limitation of ADI from Pseudomonas plecoglossicida (PpADI) lies in its pH‐dependent activity profile, its pH optimum is at 6.5. A pH shift from 6.5 to 7.5 results in an approximately 80 % drop in activity. (The pH of human plasma is 7.35 to 7.45.) In order to shift the PpADI pH optimum, a directed‐evolution protocol based on an adapted citrulline‐screening protocol in microtiter‐plate format was developed and validated. A proof of concept for ADI engineering resulted in a pH optimum of pH 7.0 and increased resistance under physiological and slightly alkaline conditions. At pH 7.4, variant M2 (K5T/D44E/H404R) is four times faster than the wild‐type PpADI and retains ∼50 % of its activity relative to its pH optimum, compared to ∼10 % in the case of the wild‐type PpADI.

A Potential Antitumor Drug (Arginine Deiminase) Reengineered for Efficient Operation under Physiological Conditions

Arginine deiminase (ADI, EC 3.5.3.6) is a potential antitumor drug for the treatment of arginine‐auxotrophic tumors such as hepatocellular carcinomas (HCCs) and melanomas, and studies on human lymphatic leukemia cell lines have confirmed that ADI has antiangiogenic activity. Recent studies showed that a combination of taxane and ADI‐PEG20, which induces caspase‐independent apoptosis, is more effective than taxane monotherapy for prostate cancer. The main limitation of ADI from Pseudomonas plecoglossicida (PpADI) and of many other ADI enzymes lies in their pH‐dependent activity profile. PpADI has a pH optimum at 6.5 and a pH shift from 6.5 to 7.5 results in an ∼80 % activity drop (the pH of human plasma is 7.35 to 7.45). In 2010, we reported a proof of concept for ADI engineering by directed evolution that resulted in variant M2 (K5T/D44E/H404R). M2 has a pH optimum of pH 7.0, a fourfold higher kcat value than the wild‐type PpADI (pH 7.4, 0.5 M phosphate buffer), and an increased Km value for substrate arginine. In our latest work, variants M5 (K5T/D38H/D44E/A128T/H404R) and M6 (K5T/D38H/D44E/A128T/E296K/H404R) were generated by directed evolution by employing PBS buffer (pH 7.4), which mimics physiological conditions. The S0.5 value of parent M3 (K5T/D44E/A128T/H404R) decreased from 2.01 to 1.48 mM (M5) and 0.81 mM (M6). The S0.5 value of M6 (0.81 mM) is lower than that of wild‐type PpADI (1.30 mM); the kcat values improved from 0.18 s−1 (wild‐type PpADI) to 17.56 s−1 (M5, 97.6‐fold) and 11.64 s−1 (M6, 64.7‐fold).

Highly efficient biosynthesis of sucrose-6-acetate with cross-linked aggregates of Lipozyme TL 100 L

As a short chain monoester, sucrose-6-acetate (S-6-a) is a key intermediate in the preparation of an eminent sweetener (sucralose). To replace the traditional multi-step chemical route for sucralose biosynthesis, enzymatic synthesis of S-6-a was investigated, using cross-linked enzyme aggregates (CLEAs) of Lipozyme TL 100 L. The optimal CLEA preparation conditions was obtained as follows: using 33.3% (v/v) PEG600 co-precipitated with additive of D-sorbierite, then cross-linking with 1.5% (v/v) glutaraldehyde at 0 °C for 4 h. As a result, the immobilized Lipozyme had high specific bioactivity (34.64 U/g) of transesterification in non-aqueous media. With these immobilized enzymes, the optimum transesterification conditions were investigated systematically, including CLEA loading, the mole ratio of vinyl acetate versus sucrose, temperature and reaction time, etc. The results showed that the highest concentration and yield of S-6-a was 49.8 g/L and 87.46%, respectively. Further experiments showed that the resulting CLEAs also had much higher operational stability than the commercial Lipozyme TLIM. The present work has paved a new path for the large-scale bioproduction of S-6-a with immobilized lipase in the future.