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The xyloglucanase gene (RmXEG12A) from Rhizomucor miehei CAU432 was successfully expressed in Pichia pastoris. The highest xyloglucanase activity of 25,700 U mL−1 was secreted using high cell density fermentation. RmXEG12A was optimally active at pH 7.0 and 65 °C, respectively. The xyloglucanase exhibited the highest specific activity towards xyloglucan (7915.5 U mg−1). RmXEG12A was subjected to hydrolyze tamarind powder to produce xyloglucan oligosaccharides with the degree of polymerization (DP) 7–9. The hydrolysis ratio of xyloglucan in tamarind powder was 89.8%. Moreover, xyloglucan oligosaccharides (2.0%, w /w) improved the water holding capacity (WHC) of yoghurt by 1.1-fold and promoted the growth of Lactobacillus bulgaricus and Streptococcus thermophiles by 2.3 and 1.6-fold, respectively. Therefore, a suitable xyloglucanase for tamarind powder hydrolysis was expressed in P. pastoris at high level and xyloglucan oligosaccharides improved the quality of yoghurt. • A xyloglucanase (RmXEG12A) from Rhizomucor miehei was expressed in P. pastoris with the activity of 25,700 U mL−1. • The hydrolysis ratio of xyloglucan in tamarind powder by RmXEG12A was up to 89.8%. • High concentration (15%, w/v) of tamarind powder was used for hydrolysis by RmXEG12A. • Xyloglucan oligosaccharides promoted the growth of starter strains in yoghurt. [ABSTRACT FROM AUTHOR]

• The β-mannanase gene (AnMan26) was codon optimized and expressed in Pichia pastoris. • High β-mannanase activity of 22,100 U mL−1 was produced through high cell density fermentation. • N-glycosylation improved the specific activity of AnMan26. • AnMan26 effectively hydrolyzed galactomannan in fenugreek gum (82.2 %). A codon-optimised β-mannanase gene from Aspergillus niger (AnMan26) was heterologously expressed in Pichia pastoris. The peak enzyme activity of 22,100 U mL−1 was achieved in a 5-L fermenter. AnMan26 was purified to homogeneity with 1.6-fold purification via Sephacryl S100 HR gel filtration. It exhibited optimal activity at pH 5.0 and 45 °C. AnMan26 was stable within the pH range 2.5–7.0 and exhibited good thermostability up to 40 °C. Moreover, it revealed highest specific activity of 2869.0 U mg−1 towards locust bean gum, and efficiently degraded various mannans to manno-oligosaccharides and hydrolysed mannotetraose and mannopentaose. Furthermore, AnMan26 hydrolysed fenugreek gum to yield partially hydrolysed fenugreek gum (PHFG) with the weight-average molecular weight of 1.8 × 103 Da. The hydrolysis ratio of galactomannan in fenugreek gum was 82.2 %. Thus, an effective β-mannanase for PHFG production was produced in this study. [ABSTRACT FROM AUTHOR]

Abstract A novel α-amylase gene (Td AmyA) with an open reading frame of 1431 bp, deducing 476 amino acids, was cloned from the thermophilic fungus Thermomyces dupontii L18. The recombinant α-amylase was successfully over-expressed in Pichia pastoris. The highest α-amylase activity of 38,314 U/mL was obtained with protein content of 28.7 mg/mL after 168 h high-cell density fermentation. Molecular mass of purified Td AmyA was 61.2 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 59.2 kDa by gel filtration. Td AmyA was a glycoprotein with 5.3% (w /w) of carbohydrate. Td AmyA exhibited maximal activity at 60 °C and pH 6.5, and was thermostable up to 55 °C within pH 4.5–10.0. It was more active towards linear starchy substrates than branched ones. The hydrolysis products were mainly comprised of maltose and maltotriose. Td AmyA produced the highest maltose content of 51.8% after 8 h hydrolysis. Thus, Td AmyA might be a candidate α-amylase for maltose syrup production. [ABSTRACT FROM AUTHOR]

An engineered β-mannanase (m Rm Man5A) from Rhizomucor miehei was successfully expressed in Pichia pastoris . Through high cell density fermentation, the expression level of m Rm Man5A reached 79,680 U mL −1 . The m Rm Man5A showed maximum activity at pH 4.5 and 65 °C, and exhibited high specific activities towards mannans. To produce manno-oligosaccharides, palm kernel cake (PKC) was pretreated by steam explosion at 200 °C for 7.5 min, and then hydrolyzed by m Rm Man5A. As a result, the total manno-oligosaccharide yield reached 34.8 g/100 g dry PKC, indicating that 80.6% of total mannan in PKC was hydrolyzed. Moreover, the kilo-scale production of manno-oligosaccharides was carried out to verify the feasibility of mass production. A total of 261.3 g manno-oligosaccharides were produced from 1.0 kg of dry PKC. An effective β-mannanase for the bioconversion of mannan-rich biomasses and an efficient method for the production of manno-oligosaccharides from PKC are provided in this paper. [ABSTRACT FROM AUTHOR]

Partially hydrolyzed guar gum (PHGG), an important supplemental dietary fiber, has been used as food ingredient in many industries. In this study, a novel β-mannanase gene ( RmMan5A ) from Rhizomucor miehei was successfully expressed in Pichia pastoris and subjected for PHGG production. Enzyme activity of fermentation supernatant reached 85,200 U mL −1 after 168 h high cell density fermentation. The purified Rm Man5A exhibited the highest enzyme activity at pH 7.0 and 65 °C. Rm Man5A was then employed for guar gum hydrolysis and PHGG obtained demonstrated a weight-average molecular weight (Mw) of 2.5 × 10 4 Da. Total dietary fiber accounted 90.6% of PHGG and 24.9% (w/w) of PHGG were identified as manno-oligosaccharides with degree of polymerization < 7. PHGG was further fractionated (F1–F4) by gradual ethanol precipitation. PHGG F1 with an Mw value of 3.6 × 10 4 Da and a mannose/galactose (M/G) ratio of 1.47 was precipitated initially, followed by PHGG F2 and F3 which showed lower Mw and higher M/G ratio. According to the structure analysis, the distribution of α- d -galactose of PHGG F1 was compact and regular, and that of other fractions was more random. A suitable β-mannanase for PHGG production and some useful information of PHGG are provided in this paper. [ABSTRACT FROM AUTHOR]

• A mutant glycoside hydrolase family 134 β-mannanase was expressed in Pichia pastoris. • m Rm Man134A showed good pH stability, thermostability, and cold-activity. • Cassia gum was used as substrate for production of manno-oligosaccharides. • Seven components were separated from the hydrolysate of cassia gum. • Prebiotic activity of manno-oligosaccharides was tested with fourteen probiotics. To produce manno-oligosaccharides from cassia gum, a mutated glycoside hydrolase family 134 β-mannanase gene (mRmMan134A) from Rhizopus microsporus var. rhizopodiformis F518 was expressed in Pichia pastoris and a high expression level (3680 U mL−1) was obtained through high cell density fermentation. m Rm Man134A exhibited maximum activity at pH 5.5 and 50 °C. It was then subjected to hydrolyze cassia gum with 70.6% of overall yield of manno-oligosaccharides. From the hydrolysate, seven components (F1–F7) were separated and identified as mannose, mannobiose, galactose, mannotriose, mannotetraose, 61-α- d -galactosyl-β- d -mannobiose, and mannopentaose, respectively. According to in vitro fermentation, the manno-oligosaccharides were able to promote the growth of three Bifidobacterium strains and six Lactobaillus strains with 3.0-fold increment in culture absorbance, and these strains preferred manno-oligosaccharides with degree of polymerization (DP) 2–3 rather than those with DP 4–5. Novel manno-oligosaccharides from cassia gum with promising prebiotic activity were provided in the present study. [ABSTRACT FROM AUTHOR]

A novel α-amylase gene (RmAmyA) from Rhizomucor miehei was cloned and expressed in Pichia pastoris. RmAmyA showed 70% amino acid identity with the α-amylase from Rhizomucor pusillus. A high α-amylase activity of 29,794.2 U/mL was found through high cell density fermentation. The molecular mass of RmAmyA was determined to be 49.9 kDa via SDS-PAGE. RmAmyA was optimally active at 75 °C and pH 6.0, and it did not require Ca2+ to improve its activity. It exhibited broad substrate specificity towards amylose, amylopectin, soluble starch, pullulan, and cyclodextrins. High level of maltose (54%, w/w) was produced after liquefied starch was hydrolysed with RmAmyA for 16 h. Moreover, the addition of RmAmyA into Chinese steamed bread resulted in 7.7% increment in the specific volume, and 17.2% and 11.5% reduction in the chewiness and hardness, respectively. These results indicate that RmAmyA might be a potential candidate for applications in the food industry. • A novel thermostable α-amylase from Rhizomucor miehei was expressed in Pichia pastoris. • High enzyme activity of 29,794.2 U/mL was obtained through high cell-density fermentation. • Liquefied starch was saccharified by this α-amylase to produce high-maltose syrup (54%). • This α-amylase could improve the quality of Chinese steamed bread and prevent staling. [ABSTRACT FROM AUTHOR]