Substrate Conversion Ratio Research Articles

ТЕСТУВАННЯ МОЖЛИВОСТІ ПЕРЕРОБКИ ЛИЧИНКАМИ HERMETIA ILLUCENS ЗАБРУДНЕНИХ МІКОТОКСИНАМИ ВІДХОДІВ АГРОПРОМИСЛОВОГО КОМПЛЕКСУ

A study of the effects on the growth, survival rate and development of H. illucens larvae contaminated with mycotoxins by-products of the agro-industrial complex is presented. For the investigated substrates, the level of substrate reduction by larvae and substrate conversion ratio in H. illucens biomass were determined. The content of the mycotoxin deoxynivalenol (DON) in protein meal produced from H. illucens larvae that grew and developed on a substrate prepared with the addition of mycotoxin contaminated corn was investigated.

2-Hydroxyisovalerate production by Klebsiella pneumoniae

2-Hydroxyisovalerate is a valuable chemical that can be used in the production of biodegradable polyesters. In nature, it was only produced at a very low level by Lactococcus lactis. 2-Ketoisovalerate is an intermediate metabolite of the branched-chain amino acid biosynthesis pathway, and Klebsiella pneumoniae ΔbudAΔldhA (Kp ΔbudAΔldhA) was a 2-ketoisovalerate producing strain. In this research, 2-hydroxyisovalerate was identified as a metabolite of Kp ΔbudAΔldhA, and its synthesis pathway was revealed. It was found that 2-ketoisovalerate and 2-hydroxyisovalerate were produced by Kp ΔbudA and Kp ΔbudAΔldhA, but not by Kp ΔbudAΔldhAΔilvD in which the 2-ketoisovalerate synthesis was blocked. budA, ldhA, and ilvD encode α-acetolactate decarboxylase, lactate dehydrogenase, and dihydroxy acid dehydratase, respectively. Thus, it was deduced that 2-hydroxyisovalerate was synthesized from 2-ketoisovalerate. Isoenzymes of ketopantoate reductase PanE, PanE2, and IlvC were suspected of being responsible for this reaction. Kinetic parameters of these enzymes were detected, and they all hold the 2-ketoisovalerate reductase activities. PanE and PanE2 use both NADH and NADPH as co-factors. While IlvC only uses NADH as a co-factor. Over-expression of panE, panE2, or ilvC in Kp ΔbudAΔldhA all enhanced the production of 2-hydroxyisovalerate. Accordingly, 2-hydroxyisovalerate levels were reduced by knocking out panE or panE2. In fed-batch fermentation, 14.41 g/L of 2-hydroxyisovalerate was produced by Kp ΔbudAΔldhA-panE, with a substrate conversion ratio of 0.13 g/g glucose.

The pyruvate decarboxylase activity of IpdC is a limitation for isobutanol production by Klebsiella pneumoniae

BackgroundKlebsiella pneumoniae contains an endogenous isobutanol synthesis pathway. The ipdC gene annotated as an indole-3-pyruvate decarboxylase (Kp-IpdC), was identified to catalyze the formation of isobutyraldehyde from 2-ketoisovalerate.ResultsCompared with 2-ketoisovalerate decarboxylase from Lactococcus lactis (KivD), a decarboxylase commonly used in artificial isobutanol synthesis pathways, Kp-IpdC has an 2.8-fold lower Km for 2-ketoisovalerate, leading to higher isobutanol production without induction. However, expression of ipdC by IPTG induction resulted in a low isobutanol titer. In vitro enzymatic reactions showed that Kp-IpdC exhibits promiscuous pyruvate decarboxylase activity, which adversely consume the available pyruvate precursor for isobutanol synthesis. To address this, we have engineered Kp-IpdC to reduce pyruvate decarboxylase activity. From computational modeling, we identified 10 amino acid residues surrounding the active site for mutagenesis. Ten designs consisting of eight single-point mutants and two double-point mutants were selected for exploration. Mutants L546W and T290L that showed only 5.1% and 22.1% of catalytic efficiency on pyruvate compared to Kp-IpdC, were then expressed in K. pneumoniae for in vivo testing. Isobutanol production by K. pneumoniae T290L was 25% higher than that of the control strain, and a final titer of 5.5 g/L isobutanol was obtained with a substrate conversion ratio of 0.16 mol/mol glucose.ConclusionsThis research provides a new way to improve the efficiency of the biological route of isobutanol production.

Blocking the 2,3-butanediol synthesis pathway of Klebsiella pneumoniae resulted in L-valine production.

Klebsiella pneumoniae is a 2,3-butanediol producing bacterium. Nevertheless, a design and construction of L-valine production strain was studied in this paper. The first step of 2,3-butanediol synthesis and branched-chain amino acid synthesis pathways share the same step of α-acetolactate synthesis from pyruvate. However, the two pathways are existing in parallel and do not interfere with each other in the wild-type strain. A knockout of budA blocked the 2,3-butanediol synthesis pathway and resulted in the L-valine production. The budA coded an α-acetolactate decarboxylase and catalyzed the acetoin formation from α-acetolactate. Furthermore, blocking the lactic acid synthesis by knocking out of ldhA, which is encoding a lactate dehydrogenase, improved the L-valine synthesis. 2-Ketoisovalerate is the precursor of L-valine, it is also an intermediate of the isobutanol synthesis pathway, while indole-3-pyruvate decarboxylase (ipdC) is responsible for isobutyraldehyde formation from 2-ketoisovalerate. Production of L-valine has been improved by knocking out of ipdC. On the other side, the ilvE, encoding a transaminase B, reversibly transfers one amino group from glutamate to α-ketoisovalerate. Overexpression of ilvE exhibited a distinct improvement of L-valine production. The brnQ encodes a branched-chain amino acid transporter, and L-valine production was further improved by disrupting brnQ. It is also revealed that weak acidic and aerobic conditions favor L-valine production. Based on these findings, L-valine production by metabolically engineered K. pneumonia was examined. In fed-batch fermentation, 22.4g/L of L-valine was produced by the engineered K. pneumoniae ΔbudA-ΔldhA-ΔipdC-ΔbrnQ-ilvE after 55h of cultivation, with a substrate conversion ratio of 0.27mol/mol glucose.

1,2-Propanediol production from glycerol via an endogenous pathway of Klebsiella pneumoniae.

Klebsiella pneumoniae is an important microorganism and is used as a cell factory for many chemicals production. When glycerol was used as the carbon source, 1,3-propanediol was the main catabolite of this bacterium. K. pneumoniae ΔtpiA lost the activity of triosephosphate isomerase and prevented glycerol catabolism through the glycolysis pathway. But this strain still utilized glycerol, and 1,2-propanediol became the main catabolite. Key enzymes of 1,2-propanediol synthesis from glycerol were investigated in detail. dhaD and gldA encoded glycerol dehydrogenases were both responsible for the conversion of glycerol to dihydroxyacetone, but overexpression of the two enzymes resulted in a decrease of 1,2-propanediol production. There are two dihydroxyacetone kinases (I and II), but the dihydroxyacetone kinase I had no contribution to dihydroxyacetone phosphate formation. Dihydroxyacetone phosphate was converted to methylglyoxal, and methylglyoxal was then reduced to lactaldehyde or hydroxyacetone and further reduced to form 1,2-propanediol. Individual overexpression of mgsA, yqhD, and fucO resulted in increased production of 1,2-propanediol, but only the combined expression of mgsA and yqhD showed a positive effect on 1,2-propanediol production. The process parameters for 1,2-propanediol production by Kp ΔtpiA-mgsA-yqhD were optimized, with pH 7.0 and agitation rate of 350rpm found to be optimal. In the fed-batch fermentation, 9.3g/L of 1,2-propanediol was produced after 144h of cultivation, and the substrate conversion ratio was 0.2g/g. This study provides an efficient way of 1,2-propanediol production from glycerol via an endogenous pathway of K. pneumoniae.Key points• 1,2-Propanediol was synthesis from glycerol by a tpiA knocked out K. pneumoniae• Overexpression of mgsA, yqhD, or fucO promote 1,2-propanediol production• 9.3g/L of 1,2-propanediol was produced in fed-batch fermentation.

Production of 2,3-dihydroxyisovalerate by Enterobacter cloacae

2,3-Dihydroxyisovalerate is an intermediate of the valine synthesis pathway. However, neither natural microorganisms nor valine producing engineered strains have been reported yet to produce this chemical. Based on the 2,3-butanediol synthesis pathway, a biological route of 2,3-dihydroxyisovalerate production was developed using a budA and ilvD disrupted Klebsiella pneumoniae strain in our previous research. We hypothesised, that other 2,3-butanediol producing bacteria could be used for 2,3-dihydroxyisovalerate production. Here a budA disrupted Enterobacter cloacae was constructed, and this strain exhibited a high 2,3-dihydroxyisovalerate producing ability. Disruption of ilvD in E. cloacae ΔbudA further increased 2,3-dihydroxyisovalerate level. The disruption of budA, encoding an acetolactate decarboxylase, resulted in the acetolactate synthesized in the 2,3-butanediol synthesis pathway to flow into the valine synthesis pathway. The additional disruption of ilvD, encoding a dihydroxy acid dehydratase, prevented the 2,3-dihydroxyisovalerate to be further metabolized in the valine synthesis pathway. Thus, the disruption of both budA and ilvD in 2,3-butanediol producing strains might be an universal strategy for 2,3-dihydroxyisovalerate accumulation. After optimization of the medium components and culture parameters 31.2 g/L of 2,3-dihydroxyisovalerate was obtained with a productivity of 0.41 g/L h and a substrate conversion ratio of 0.56 mol/mol glucose in a fed-batch fermentation. This approach provides an economic way for 2,3-dihydroxyisovalerate production.

Ethylene glycol and glycolic acid production by wild-type Escherichia coli.

Ethylene glycol and glycolic acid are bulk chemicals with a broad range of applications. The ethylene glycol and glycolic acid biosynthesis pathways have been produced by microorganisms and used as a biological route for their production. Unlike the methods that use xylose or glucose as carbon sources, xylonic acid was used as a carbon source to produce ethylene glycol and glycolic acid in this study. Amounts of 4.2 g/L of ethylene glycol and 0.7 g/L of glycolic acid were produced by a wild-type Escherichia coli W3110 within 10 H of cultivation with a substrate conversion ratio of 0.5 mol/mol. Furthermore, E. coli strains that produce solely ethylene glycol or glycolic acid were constructed. 10.3 g/L of glycolic acid was produced by E. coli ΔyqhD+aldA, and the achieved conversion ratio was 0.56 mol/mol. Similarly, the E. coli ΔaldA+yqhD produced 8.0 g/L of ethylene glycol with a conversion ratio of 0.71 mol/mol. Ethylene glycol and glycolic acid production by E. coli on xylonic acid as a carbon source provides new information on the biosynthesis pathway of these products and opens a novel way of biomass utilization.

Water-in-oil microcompartments for the study of biomimetic drug metabolism

Microcompartments in the form of water-in-oil droplets have been utilized to construct artificial cells and simulate human body environment. However, the performance of subcellular structure involved metabolism in emulsion droplets has not been explored, and the underlying mechanism is still being elucidated. In this work, drug metabolism is presented on the basis of great amounts of microcompartments formed of picoliter-volume droplets with different radius (R), using a commercial four-way valve as a droplet generator. A model substrate, phenacetin, and its metabolite, paracetamol, are quantitatively analyzed by liquid-chromatography (LC) tandem mass spectrometry (MS/MS), and the reaction kinetics is characterized. In microdroplets of varying size (R = 18, 27, 42, and 51 μm, respectively), both conversion ratio and reaction rate constant of the metabolism are influenced in different degree. For instance, the substrate conversion ratio after 60 min of incubation in R = 27 μm droplets improves from 15% to 42%, and the reaction rate constant improves nearly five-fold, compared to that in bulk phase. The influence of microcompartment size on metabolism rate is further explored by simulation using a diffusion-reaction model. The droplet-based strategy is rapid, accurate and cost-efficient, fitting especially into biomimetic metabolism studies.

Removal of heavy oil using rhinoceros beetle, Oryctes rhinoceros

The remnant of heavy oil, often expressed as total petroleum hydrocarbons (TPHs), in soil and/or sludge is commonly encountered by and often ruins the habitat of some indigenous insects. Recent studies indicate that some of the insects could stand the impact and some not just survived but might serve as an oil degrader. The potential of using indigenous insects in treating oil pollution is of interests in this study. In Asia, the potential oil-exposed rhinoceros beetle, Oryctes rhinoceros, was studied herein due to two major interests: one is its tolerance to heavy oil, and the other its ability to remove the oil. Two sets of the experiments spiked with different amounts of heavy oil were conducted in series, and the results of the larval mortality rate, growth rate, feces production, substrate conversion ratio and percent of oil removal were monitored as performance indicators during these treatments. The obtained results showed that the ingestion of heavy oil at a 5000 mg/kg level significantly deteriorated the survival rate (37% left) of the tested larvae, yet oil-experienced larvae could survive in up to 10,000 mg/kg of heavy oil (100% survived). As for the heavy oil removal via larval ingestion, at a 10,000 mg/kg level of oil in the substrate, the percent of weekly removal of heavy oil by larva ingestion was consistent throughout the second run at 56.4 ± 5.6%. These results demonstrated the potential use of beetle larvae as a bioreactor in pollutant removal.

Continuous hydrogen production by dark and photo co-fermentation using a tubular multi-cycle bio-reactor with Paulownia biomass

Bio-hydrogen production from lignocellulosic biomass has shown significant potential for helping to meet future energy demand. In the present work, we have utilized the Paulownia lignocellulosic biomass as feedstock for hydrogen production which has great potential for sustainable supply over the year due to its high growth rate. A dark and photo co-fermentative mechanism was adopted in a newly proposed tubular multi-cycle bioreactor and a 30-day continuous hydrogen production trial was conducted in the reactor. The key factors such as hydraulic retention time and organic loading rate associated with hydrogen production and substrate degradation were regulated to optimize the bioprocess. Our experimental findings show that with increase in hydraulic retention time and organic loading rate, hydrogen yield decreases and increases respectively. We have observed hydrogen yield, hydrogen concentration and substrate conversion ratio of 0.54 L/h, 53.84%, and 89.93% at optimized parameters of hydraulic retention time of 40 h, OLR of 1.0 g/L h, and light–dark interval time of 1:4 respectively. Our experimental finds may help to further explore the hydrogen production potential from lignocellulosic biomass of Paulownia.

Upgrading liquor-making wastewater into medium chain fatty acid: Insights into co-electron donors, key microflora, and energy harvest

Ethanol and lactate are considered suitable electron donors (EDs) for chain elongation (CE); however, their respective shortcomings still limit the substrate conversion ratio and medium chain fatty acid (MCFA) production. To address this limitation, different EDs and electron acceptors (EAs) were combined to compare their CE performances, and to investigate whether the combination of ethanol and lactate could further enhance the MCFA production based on the complementary characteristics of ethanol and lactate. The results verified, for the first time, ethanol and lactate as the co-EDs formed a cooperative relationship to largely promote the conversion of substrates into MCFA. The co-EDs of ethanol and lactate stimulated the transformation of dispersive lactate-carbon flux from the competing acrylate pathway into n-heptylate. Additionally, the coexisting by-products (H2 and CO2) from ethanol and lactate also contributed to the supererogatory MCFA generation. The key microbial taxa that distinguished the co-EDs from their single action were the preponderant species from class Negativicutes and family Ruminococcaceae. In addition, the co-EAs of acetate, n-butyrate, and n-caproate also promoted MCFA generation. Low concentration of n-caproate could be directly elongated into n-caprylate, while n-caproate concentration exceeding the toxic limit was unsuitable as an EA. This research provided a guide for substrate selection and collocation for CE technology. Chinese liquor-making wastewater (CLMW) was subsequently used as a substrate for MCFA production since it contains abundant lactate, ethanol, and short-chain fatty acids. In this study, a MCFA selectivity of 80.34 ± 5.26%, a slightly higher selectivity which is in the range of previously reported ones, was obtained. This study paves a way for the sustainable development of Chinese liquor industry by recycling the high-output CLMW into MCFA.

The Role of the Pyruvate Acetyl-CoA Switch in the Production of 1,3-Propanediol by Klebsiella pneumoniae.

Pyruvate dehydrogenase-complex (AcoABCD) and pyruvate formate-lyase (PFL) are two pathways responsible for synthesis of acetyl-CoA from pyruvate (pyruvate acetyl-CoA switch). The two pathways were individually deleted in Klebsiella pneumoniae, and the role of the pyruvate acetyl-CoA switch in 1,3-propanediol production was investigated. Fermentation results showed that the two pathways were both active in the wild-type strain. Acetyl-CoA formation between the two pathways was equal in the wild-type strain. The pflB mutant produced high level of lactic acid, and deletion of ldhA eliminated lactic acid synthesis. The conversion ratio of glycerol to 1,3-propanediol in the pflB-ldhA mutant reached 0.541g/g, which was 9.4% higher than that of the wild-type strain. However, the productivity of 1,3-propanediol was decreased in the pflB-ldhA mutant. In contrast, the productivity of 1,3-propanediol was increased by 19% in the acoABCD mutant, with the disadvantage of lower substrate conversion ratio. Regulating the pyruvate acetyl-CoA switch presents a novel way to improve the conversion ratio or productivity of 1,3-propanediol produced by K. pneumoniae.

Ethanolic Fermentation Efficiency of Seaweed Solid Waste hydrolysates by Saccharomyces cerevisiae

The objective of this research are to determine the Saccharification and fermentation efficiency of seaweed solid waste hydrolysate by S. cerevisiae in anaerobic condition. The optimum saccharification yield of acid pretreated waste (40.93+1.72%) was obtained after 48 h with a saccharification rate of 0.51±0.02 g/L/h. Higher yield was showed by NaOH pretreated waste (67.29+1.24%) at 24 hours with a saccharification rate of 0.81±0.06 g/L/h. . Fermentation of enzymatic hydrolysates of acid and base pretreated samples with S. cerevisiae produced maximum ethanol 7.52±0.24 g/L and 14.5+0.54 g/L respectively after 72 hours fermentation. Maximum ethanol yield of 0.31±0.03 g/g and 0.40+0.02 g/g sugar respectively for acid and base pretreated samples. The ethanol yields showing that base pretreated sample was producing higher conversion ratio of substrate (80% of theoretical yield) compared to acid pretreated sample (62% of theoretical yield).

Combination of phenylpyruvic acid (PPA) pathway engineering and molecular engineering of L-amino acid deaminase improves PPA production with an Escherichia coli whole-cell biocatalyst.

In our previous study, we produced phenylpyruvic acid (PPA) in one step from L-phenylalanine by using an Escherichia coli whole-cell biocatalyst expressing an L-amino acid deaminase (L-AAD) from Proteus mirabilis KCTC2566. However, the PPA titer was low due to the degradation of PPA and low substrate specificity of L-AAD. In this study, metabolic engineering of the L-phenylalanine degradation pathway in E. coli and protein engineering of L-AAD from P. mirabilis were performed to improve the PPA titer. First, three aminotransferase genes were knocked out to block PPA degradation, which increased the PPA titer from 3.3 ± 0.2 to 3.9 ± 0.1 g/L and the substrate conversion ratio to 97.5 %. Next, L-AAD was engineered via error-prone polymerase chain reaction, followed by site-saturation mutation to improve its catalytic performance. The triple mutant D165K/F263M/L336M produced the highest PPA titer of 10.0 ± 0.4 g/L, with a substrate conversion ratio of 100 %, which was 3.0 times that of wild-type L-AAD. Comparative kinetics analysis showed that compared with wild-type L-AAD, the triple mutant had higher substrate-binding affinity and catalytic efficiency. Finally, an optimal fed-batch biotransformation process was developed to achieve a maximal PPA titer of 21 ± 1.8 g/L within 8 h. This study developed a robust whole-cell E. coli biocatalyst for PPA production by integrating metabolic and protein engineering, strategies that may be useful for the construction of other biotransformation biocatalysts.

R-acetoin accumulation and dissimilation in Klebsiella pneumoniae.

Klebsiella pneumoniae is a 2,3-butanediol producer, and R-acetoin is an intermediate of 2,3-butanediol production. R-acetoin accumulation and dissimilation in K. pneumoniae was studied here. A budC mutant, which has lost 2,3-butanediol dehydrogenase activity, accumulated high levels of R-acetoin in culture broth. However, after glucose was exhausted, the accumulated R-acetoin could be reused by the cells as a carbon source. Acetoin dehydrogenase enzyme system, encoded by acoABCD, was responsible for R-acetoin dissimilation. acoABCD mutants lost the ability to grow on acetoin as the sole carbon source, and the acetoin accumulated could not be dissimilated. However, in the presence of another carbon source, the acetoin accumulated in broth of acoABCD mutants was converted to 2,3-butanediol. Parameters of R-acetoin production by budC mutants were optimized in batch culture. Aerobic culture and mildly acidic conditions (pH 6-6.5) favored R-acetoin accumulation. At the optimized conditions, in fed-batch fermentation, 62.3g/L R-acetoin was produced by budC and acoABCD double mutant in 57h culture, with an optical purity of 98.0%, and a substrate conversion ratio of 28.7%.

Highly efficient dynamic kinetic resolution of secondary aromatic alcohols at low temperature using a low-cost sulfonated sepiolite as racemization catalyst

A highly efficient dynamic kinetic resolution system for secondary aromatic alcohol using low-cost sulfonated sepiolite as a racemization catalyst has been developed. The system operates at 25°C, achieves good eep (>99%) and substrate conversion ratio (>99%), is applicable to a variety of substrates and can be reused more than 10 times.

A high throughput method to screen companion bacterium for 2-keto-l-gulonic acid biosynthesis by co-culturing Ketogulonicigenium vulgare

Bacillus megaterium is widely used as companion bacterium in the two-step biosynthesis of 2-keto-l-gulonic acid (2-KLG) by Ketogulonicigenium vulgare. To screen efficiently target companion strains from large numbers of random mutants, a screen method based on spectrophotometry and 24-well microtiter plates was developed and validated on an integrated library of 450 transposon random insertional mutants and two sporulation-defective mutants. The co-culture processes were classified into three groups (low, intermediate and high performance) by K-mean clustering analysis. In addition, mutant m71 was successfully screened out from the library. The substrate conversion ratio of m71 and K. vulgare co-culture process after 72h was decreased by about 38% compared with that of the wild-type co-culture process in 750ml flasks. These results indicated that the proposed high throughput method is feasible for screening target companions for the co-culture process of 2-KLG biosynthesis.

Eryngase: a Pleurotus eryngii aminopeptidase exhibiting peptide bond formation activity

An aminopeptidase that has peptide bond formation activity was identified in the cell-free extract of carpophore of Pleurotus eryngii. The enzyme, redesignated as eryngase, was purified for homogeneity and characterized. Eryngase had a molecular mass of approximately 79 kDa. It showed somewhat high stability with respect to temperature and pH; it was inhibited by iodoacetate. Among hydrolytic activities toward aminoacyl-p-nitroanilides (aminoacyl-pNAs), eryngase mainly hydrolyzed hydrophobic L-aminoacyl-pNAs and exhibited little activity toward D-Ala-pNA and D-Leu-pNA. In terms of peptide bond formation activity, eryngase used various aminoacyl derivatives as acyl donors and acceptors. The products were all dipeptidyl derivatives. Investigation of time dependence on peptide synthesis revealed that some peptides that are not recognized as substrates for hydrolytic activity of eryngase could become good targets for synthesis. Furthermore, eryngase has produced opioid dipeptides--L-kyotorphin (L-Tyr-L-Arg) and D-kyotorphin (L-Tyr-D-Arg)--using L-Tyr-NH(2) and D- and L-Arg-methyl ester respectively as an acyl donor and acceptor. Yield evaluation of kyotorphin synthesis indicated that the conversion ratio of substrate to kyotorphin was moderate: the value was estimated as greater than 20%.

The Aminolysis Reaction of Streptomyces S9 Aminopeptidase Promotes the Synthesis of Diverse Prolyl Dipeptides

Prolyl dipeptide synthesis by S9 aminopeptidase from Streptomyces thermocyaneoviolaceus (S9AP-St) has been demonstrated. In the synthesis, S9AP-St preferentially used l-Pro-OBzl as the acyl donor, yielding synthesized dipeptides having an l-Pro-Xaa structure. In addition, S9AP-St showed broad specificity toward the acyl acceptor. Furthermore, S9AP-St produced cyclo (l-Pro-l-His) with a conversion ratio of substrate to cyclo (l-Pro-l-His) higher than 40%.

Design and operation of an integrated membrane reactor for enzymatic cellulose hydrolysis

A purposely built membrane reactor with combined reaction and separation zone in a single piece of equipment was tested for heterogeneous enzymatic hydrolysis of a hardwood derived cellulosic material. The design and operation features a process of simultaneous reaction and in situ removal of products (reducing sugars) in order to reduce the effect of product inhibition on the cellulase enzyme. The integrated reactor system differs from the hybrid integration of reaction-separation system where the reaction takes place in a stirred reaction vessel and the separation of the product is carried out in a separate cross-flow membrane filtration unit. Various operating strategies of semi-continuous and continuous product removal and substrate feeding were examined and optimised in respect to their effect on reducing product inhibition and increasing reactor productivity and product yield. The effect of total enzyme concentration and possible enzyme deactivation by shear stress were also investigated. The findings from examining the performance of a well understood enzymatic system in a new integrated reactor system would facilitate the understanding and development of system integration and process intensification in combined reaction-separation process which may alleviate common problems (product inhibition, inefficient interfacial mass transfer, slow kinetics and low reactor productivity) in heterogeneous enzyme catalysis systems. The cellulose substrate conversion ratio was 53% in the continuous operation of reaction with simultaneous product removal. This compares favourably with 35% substrate conversion in traditional batch operations. The improvement in overall reaction rate through adopting an in situ product removal strategy was rather limited, mainly because of the inherent slow reaction kinetics of the heterogeneous cellulose hydrolysis and the complex interfacial interactions between the cellulase enzymes and the solid cellulose substrate.