Cross-linked cyclodextrin glucanotransferase aggregates from Bacillus lehensis G1 for cyclodextrin production: Molecular modeling, developmental, physicochemical, kinetic and thermodynamic properties

Type of cross-linking agents influence the stability and active cross-linked enzyme aggregates (CLEA) immobilization. The information of molecular interaction between enzyme-cross linker is not well explored thus screening wide numbers of cross-linker is crucial in CLEA development. This study combined the molecular modeling and experimental optimization to investigate the influences of different cross-linking agents in developing CLEA of cyclodextrin glucanotranferase G1 (CGTase G1) for cyclodextrins (CDs) synthesis. Seven types of cross-linkers were tested and CGTase G1 cross-linked with chitosan (CS-CGTG1-CLEA) displayed the highest activity recovery (84.6 ± 0.26%), aligning with its highest binding affinity, radius of gyration and flexibility through in-silico analysis towards CGTase G1. CS-CGTG1-CLEA was characterized and showed a longer half-life (30.06 ± 1.51 min) and retained a greater thermal stability (52.73 ± 0.93%) after 30 min incubation at optimal conditions compared to free enzyme (10.30 ± 1.34 min and 5.51 ± 2.10% respectively). CS-CGTG1-CLEA improved CDs production by 33% and yielded cumulative of 52.62 g/L CDs after five cycles for 2 h of reaction. This study reveals that abundant of hydroxyl group on chitosan interacted with CGTase G1 surface amino acid residues to form strong and stable CLEA thus can be a promising biocatalyst in CDs production

Bioconversion of starch to maltooligosaccharides (MOS) by the reaction of maltogenic amylase

Maltogenic amylase is one of the significant enzymes in oligosaccharides synthesis. Its ability to utilise multiple substrates and catalyse hydrolysis and transglycosylation reactions simultaneously makes it a unique biocatalyst. The catalysis could be exploited in many ways to obtain oligosaccharides of different lengths and various modified sugars. Nonetheless, one of the major drawbacks of substrate hydrolysis to produce oligosaccharides is the low production of MOS with higher degree of polymerisation. To address this issue, reaction parameter optimisation was performed via one-factor-at-a-time (OFAT) approach on the production of MOS from soluble starch hydrolysis using maltogenic amylase from Bacillus lehensis G1 (MAG1). Optimisation of MAG1 loading, soluble starch loading, temperature, time and pH resulted in the production of 84.87 mg/g MOS with polymerisation degree of 3 to 7 compared to that of 51.60 mg/g obtained before the optimisation process, which recorded 1.64-fold increment. Among all parameters, soluble starch loading gave the most significant impact on the MOS production as the reaction equilibrium is highly affected by substrate concentration. The occurrence of MOS with polymerisation degree of 4 and above, which resulted from starch hydrolysis further confirms the endo-type of MAG1. Because starch is an abundant and inexpensive source of carbohydrate in the world, this study provides a cost-effective MOS production process which is highly relevant for industry

Biochemical characterisation and structure determination of a novel cold-active Proline iminopeptidase from the Psychrophilic yeast, Glaciozyma antarctica PI12

Microbial proteases constitute one of the most important groups of industrially relevant enzymes. Proline iminopeptidases (PIPs) that specifically release amino-terminal proline from peptides are of major interest for applications in food biotechnology. Proline iminopeptidase has been extensively characterised in bacteria and filamentous fungi. However, no similar reports exist for yeasts. In this study, a protease gene from Glaciozyma antarctica designated as GaPIP was cloned and overexpressed in Escherichia coli. Sequence analyses of the gene revealed a 960 bp open reading frame encoding a 319 amino acid protein (35,406 Da). The purified recombinant GaPIP showed a specific activity of 3561 Umg−1 towards L-proline-p-nitroanilide, confirming its identity as a proline iminopeptidase. GaPIP is a cold-active enzyme with an optimum activity of 30◦ C at pH 7.0. The enzyme is stable between pH 7.0 and 8.0 and able to retain its activity at 10–30◦ C. Although GaPIP is a serine protease, only 25% inhibition by the serine protease inhibitor, phenylmethanesulfonylfluoride (PMSF) was recorded. This enzyme is strongly inhibited by the presence of EDTA, suggesting that it is a metalloenzyme. The dimeric structure of GaPIP was determined at a resolution of 2.4 Å. To date, GaPIP is the first characterised PIP from yeasts and the structure of GaPIP is the first structure for PIP from eukaryotes

Metabolic pathway modification for production of xylitol from glucose in escherichia coli

Glucose is a cheap and readily available substrate for production of large-scale chemicals. Synthesis of xylitol, a high demand chemical in global market is currently done by using xylose, which contributes to its high operational cost. Studies on production of xylitol from glucose have explored several approaches, from sequentia l fermentation to multiple and single gene expression. Xylitol-5-phosphate dehydrogenase (XPDH), is an enzyme that enables conversion of glucose to xylitol in a single step fermentation. This study explores conversion of xylitol from glucose in E. coli by the expression of xpdh from Clostridium difficile with modifications in metabolic pathways to enhance xylitol production. The xpdh gene was carried by pACYC-Duet-1 expression vector and induced by the addition of IPTG. Initial screening of E. coli expressing xpdh (NA116) was done by shake-flask fermentation for 24 hours and its metabolites were analyzed by HPLC. NA116 was able to produce 0.273 g/L xylitol from 4.33 g/L consumed glucose in 24 hours. Further metabolic pathway modification to eliminate competing pathways yielded four mutants, NA207 (∆rpiA), NA208 (∆rpiB), NA209 (∆pgi) and NA211 (∆rpi∆Apgi). Screening of mutants for xylitol production showed that highest xylitol production from glucose was achieved by NA211 with almost double the amount of the original strain, 0.585 g/L. This showed successful xylitol conversion from glucose in a single fermentation in E. coli with improved yield through metabolic pathway modification

Protein surface engineering and interaction studies of maltogenic amylase towards improved enzyme immobilisation

A combined strategy of computational, protein engineering and cross-linked enzyme aggregates (CLEAs) approaches was performed on Bacillus lehensis G1 maltogenic amylase (Mag1) to investigate the preferred amino acids and orientation of the cross-linker in constructing stable and efficient biocatalyst. From the computational analysis, Mag1 exhibited the highest binding affinity towards chitosan (−7.5 kcal/mol) and favours having interactions with aspartic acid whereas glutaraldehyde was the least favoured (−3.4 kcal/mol) and has preferences for lysine. A total of eight Mag1 variants were constructed with either Asp or Lys substitutions on different secondary structures surface. Mutant Mag1-mDh exhibited the highest recovery activity (82.3%) in comparison to other Mag1 variants. Mutants-CLEAs exhibited higher thermal stability (20–30% activity) at 80 °C whilst Mag1-CLEAs could only retain 9% of activity at the same temperature. Reusability analysis revealed that mutants-CLEAs can be recovered up to 8 cycles whereas Mag1-CLEAs activity could only be retained for up to 6 cycles. Thus, it is evident that amino acids on the enzyme's surface play a crucial role in the construction of highly stable, efficient and recyclable CLEAs. This demonstrates the necessity to determine the preferential amino acid by the cross-linkers in advance to facilitate CLEAs immobilisation for designing efficient biocatalysts

Protein engineering of GH11 xylanase from Aspergillus fumigatus RT-1 for catalytic efficiency improvement on kenaf biomass hydrolysis

Enzyme hydrolysis faces a bottleneck due to the recalcitrance of the lignocellulose biomass. The protein engineering of GH11 xylanase from Aspergillus fumigatus RT-1 was performed near the active site and at the N-terminal region to improve its catalytic efficiency towards pretreated kenaf (Hibiscus cannabinus) hydrolysis. Five mutants were constructed by combined approaches of error-prone PCR, site-saturation and site-directed mutagenesis. The double mutant c168 t/Q192H showed the most effective hydrolysis reaction with a 13.9-fold increase in catalytic efficiency, followed by mutants Y7L and c168 t/Q192 H/Y7L with a 1.6-fold increase, respectively. The enhanced catalytic efficiency evoked an increase in sugar yield of up to 28% from pretreated kenaf. In addition, mutant c168 t/Q192 H/Y7L improved the thermostability at higher temperature and acid stability. This finding shows that mutations at distances less than 15 Å from the active site and at putative secondary binding sites affect xylanase catalytic efficiency towards insoluble substrates hydrolysis

Crystal structure of fuculose aldolase from the Antarctic psychrophilic yeast Glaciozyma antarctica PI12

Fuculose-1-phosphate aldolase (FucA) catalyses the reversible cleavage of l-fuculose 1-phosphate to dihydroxyacetone phosphate (DHAP) and l-lactaldehyde. This enzyme from mesophiles and thermophiles has been extensively studied; however, there is no report on this enzyme from a psychrophile. In this study, the gene encoding FucA from Glaciozyma antarctica PI12 (GaFucA) was cloned and the enzyme was overexpressed in Escherichia coli, purified and crystallized. The tetrameric structure of GaFucA was determined to 1.34 Å resolution. The overall architecture of GaFucA and its catalytically essential histidine triad are highly conserved among other fuculose aldolases. Comparisons of structural features between GaFucA and its mesophilic and thermophilic homologues revealed that the enzyme has typical psychrophilic attributes, indicated by the presence of a high number of nonpolar residues at the surface and a lower number of arginine residues

Functional characterisation and product specificity of Endo-β-1,3-glucanase from alkalophilic bacterium, Bacillus lehensis G1

Endo-β-1,3-glucanase from alkalophilic bacterium, Bacillus lehensis G1 (Blg32) composed of 284 amino acids with a predicted molecular mass of 31.6 kDa is expressed in Escherichia coli and purified to homogeneity. Herein, Blg32 characteristics, substrates and product specificity as well as structural traits that might be involved in the production of sugar molecules are analysed. This enzyme functions optimally at the temperature of 70 °C, pH value of 8.0 with its catalytic activity strongly enhanced by Mn2+. Remarkably, the purified enzyme is highly stable in high temperature and alkaline conditions. It exhibits the highest activity on laminarin (376.73 U/mg) followed by curdlan and yeast β-glucan. Blg32 activity increased by 62% towards soluble substrate (laminarin) compared to insoluble substrate (curdlan). Hydrolytic products of laminarin were oligosaccharides with degree of polymerisation (DP) of 1 to 5 with the main product being laminaritriose (DP3). This suggests that the active site of Blg32 could recognise up to five glucose units. High concentration of Blg32 mainly produces glucose whilst low concentration of Blg32 yields oligosaccharides with different DP (predominantly DP3). A theoretical structural model of Blg32 was constructed and structural analysis revealed that Trp156 is involved in multiple hydrophobic stacking interactions. The amino acid was predicted to participate in substrate recognition and binding. It was also exhibited that catalytic groove of Blg32 has a narrow angle, thus limiting the substrate binding reaction. All these properties and knowledge of the subsites are suggested to be related to the possible mode of action of how Blg32 produces glucooligosaccharides